Use of microfluidic systems in the detection of target analytes using microsphere arrays

ABSTRACT

The invention relates generally to methods and apparatus for conducting analyses, particularly microfluidic devices for the detection of target analytes.

This application is a continuation-in-part of U.S. application Ser. No.09/990,890, filed Nov. 21, 2001 (pending), which claims the benefit ofU.S. Application Ser. No. 60/252,227, filed Nov. 21, 2000 (abandoned),both of which are expressly incorporated herein by reference. Thisapplication also is a continuation-in-part of U.S. application Ser. No.09/979,236, filed Apr. 15, 2002 (pending), which is a 371 ofPCT/US00/13942, filed May 22, 2000 claiming priority to U.S. applicationSer. No. 09/316,154, filed May 21, 1999 (abandoned), all of which areexpressly incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates generally to methods and apparatus for conductinganalyses, particularly microfluidic devices for the detection of targetanalytes.

BACKGROUND OF THE INVENTION

There are a number of assays and sensors for the detection of thepresence and/or concentration of specific substances in fluids andgases. Many of these rely on specific ligand/antiligand reactions as themechanism of detection. That is, pairs of substances (i.e. the bindingpairs or ligand/antiligands) are known to bind to each other, whilebinding little or not at all to other substances. This has been thefocus of a number of techniques that utilize these binding pairs for thedetection of the complexes. These generally are done by labeling onecomponent of the complex in some way, so as to make the entire complexdetectable, using, for example, radioisotopes, fluorescent and otheroptically active molecules, enzymes, etc.

One type of sensor that is showing particular promise is based onmicrospheres or beads that are distributed on a substrate at discretesites. Each bead contains a chemical functionality, such as a bindingpartner, that can be used to detect the presence of a target analyte.The beads are put down randomly and then a variety of decoding schemesare used to elucidate the location and chemical functionality at eachsite. See for example PCT US98/21193, PCT US99/04473; PCT US98/05025 andPCT US98/09163.

There is a significant trend to reduce the size of these sensors, bothfor sensitivity and to reduce reagent costs. Thus, a number ofmicrofluidic devices have been developed, generally comprising a solidsupport with microchannels, utilizing a number of different wells,pumps, reaction chambers, and the like. See for example EP 0637996 B1;EP 0637998 B1; WO96/39260; WO97/16835; WO98/13683; WO97/16561;WO97/43629; WO96/39252; WO96/15576; WO96/15450; WO97/37755; andWO97/27324; and U.S. Pat. Nos. 5,304,487; 5,071,531; 5,061,336;5,747,169; 5,296,375; 5,110,745; 5,587,128; 5,498,392; 5,643,738;5,750,015; 5,726,026; 5,35,358; 5,126,022; 5,770,029; 5,631,337;5,569,364; 5,135,627; 5,632,876; 5,593,838; 5,585,069; 5,637,469;5,486,335; 5,755,942; 5,681,484; and 5,603,351.

Thus, there is a need for a microfluidic biosensor that is both smalland high density, that can be used in a high throughput manner.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides microfluidic devices for the detection of a target analyte in asample. The devices comprise a solid support that has any number ofmodules, including a sample inlet port and at least one sample handlingwell comprising a well inlet port and a well outlet port. The devicegenerally further comprises a first microchannel to allow fluid contactbetween the sample inlet port and the sample handling well. The devicealso comprises a detection module comprising a substrate with a surfacecomprising discrete sites, and a population of microspheres comprisingat least a first and a second subpopulation, wherein each subpopulationcomprises a bioactive agent. The microspheres are distributed on saidsurface. The detection module also comprises a detection inlet port toreceive the sample. The device also comprises a second microchannel toallow fluid contact between the sample handling well and the detectioninlet port.

In addition the invention provides a method of assembling a detector ina microfluidic device. The method includes providing a microfluidicdevice comprising a first microchannel to allow fluid contact between asample inlet port and a sample handling well, a second microchannel toallow fluid contact between said sample handling well and a detectioninlet port, and a detection module comprising a substrate with a surfacecomprising discrete sites. The method further includes flowing a fluidacross the substrate. The fluid comprises a population of microspherescomprising at least a first and a second subpopulation, wherein eachsubpopulation comprises a bioactive agent, whereby the beads flow acrossthe discrete sites, and are deposited randomly in the discrete sites.The method additionally includes reversing the flow of the fluid.

In addition the invention provides a method of assembling a detector ina microfluidic device. The method includes providing a microfluidicdevice comprising a plurality of first micro channels, and a populationof microspheres in microchannels. The device further includes areceiving chamber connected to said microchannels. The method furtherincludes flowing said microspheres through said microchannels into saidreceiving chamber.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically depicts improved signal intensity with vibration ofthe chip during hybridization.

FIG. 2 depicts a microchannel tree structure for on-chip beaddistribution. Each reservoir can be filled with a different beadsolution.

FIG. 3 depicts a configuration of a bead-counting region built into achannel of a tree structure or network.

FIG. 4 depicts bead loading by sequentially reversing the direction offlow over the microarray until the wells are filled.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides microfluidic cassettes or devices that can beused to effect a number of manipulations on a sample to ultimatelyresult in target analyte detection or quantification. Thesemanipulations can include cell handling (cell concentration, cell lysis,cell removal, cell separation, etc.), separation of the desired targetanalyte from other sample components, chemical or enzymatic reactions onthe target analyte, detection of the target analyte, etc. The devices ofthe invention can include one or more wells for sample manipulation,waste or reagents; microchannels to and between these wells, includingmicrochannels containing electrophoretic separation matrices; valves tocontrol fluid movement; on-chip pumps such as electroosmotic,electrohydrodynamic, or electrokinetic pumps; and detection systemscomprising bead arrays, as is more fully described below. The devices ofthe invention can be configured to manipulate one or multiple samples oranalytes.

The microfluidic devices of the invention are used to detect targetanalytes in samples. By “target analyte” or “analyte” or grammaticalequivalents herein is meant any molecule, compound or particle to bedetected. As outlined below, target analytes preferably bind to bindingligands, as is more fully described above. As will be appreciated bythose in the art, a large number of analytes may be detected using thepresent methods; basically, any target analyte for which a bindingligand, described herein, may be detected using the methods of theinvention.

Suitable analytes include organic and inorganic molecules, includingbiomolecules. In a preferred embodiment, the analyte may be anenvironmental pollutant (including pesticides, insecticides, toxins,etc.); a chemical (including solvents, polymers, organic materials,etc.); therapeutic molecules (including therapeutic and abused drugs,antibiotics, etc.); biomolecules (including hormones, cytokines,proteins, lipids, carbohydrates, cellular membrane antigens andreceptors (neural, hormonal, nutrient, and cell surface receptors) ortheir ligands, etc); whole cells (including procaryotic (such aspathogenic bacteria) and eukaryotic cells, including mammalian tumorcells); viruses (including retroviruses, herpesviruses, adenoviruses,lentiviruses, etc.); and spores; etc. Particularly preferred analytesare environmental pollutants; nucleic acids; proteins (includingenzymes, antibodies, antigens, growth factors, cytokines, etc);therapeutic and abused drugs; cells; and viruses.

In a preferred embodiment, the target analyte is a nucleic acid. By“nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al.,J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press), and peptide nucleic acid backbones and linkages (seeEgholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed.Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al.,Nature 380:207 (1996), all of which are incorporated by reference).Other analog nucleic acids include those with positive backbones (Denpcyet al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones(U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423(1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsingeret al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogsare described in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of labels or to increase the stability and half-life ofsuch molecules in physiological environments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made.Alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.

Particularly preferred are peptide nucleic acids (PNA) which includespeptide nucleic acid analogs. These backbones are substantiallynon-ionic under neutral conditions, in contrast to the highly chargedphosphodiester backbone of naturally occurring nucleic acids. Thisresults in two advantages. First, the PNA backbone exhibits improvedhybridization kinetics. PNAs have larger changes in the meltingtemperature (Tm) for mismatched versus perfectly matched basepairs. DNAand RNA typically exhibit a 2-4° C. drop in Tm for an internal mismatch.With the non-ionic PNA backbone, the drop is closer to 7-9° C. Thisallows for better detection of mismatches. Similarly, due to theirnon-ionic nature, hybridization of the bases attached to these backbonesis relatively insensitive to salt concentration.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc. As used herein, the term“nucleoside” includes nucleotides and nucleoside and nucleotide analogs,and modified nucleosides such as amino modified nucleosides. Inaddition, “nucleoside” includes non-naturally occurring analogstructures. Thus for example the individual units of a peptide nucleicacid, each containing a base, are referred to herein as a nucleoside.

In a preferred embodiment, the present invention provides methods ofdetecting target nucleic acids. By “target nucleic acid” or “targetsequence” or grammatical equivalents herein means a nucleic acidsequence on a single strand of nucleic acid. The target sequence may bea portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNAincluding mRNA and rRNA, or others. It may be any length, with theunderstanding that longer sequences are more specific. In someembodiments, it may be desirable to fragment or cleave the samplenucleic acid into fragments of 20 to 10,000 basepairs, with fragments ofroughly 500 basepairs being preferred in some embodiments. Forhybridization purposes, smaller fragments are generally preferred. Aswill be appreciated by those in the art, the complementary targetsequence may take many forms. For example, it may be contained within alarger nucleic acid sequence, i.e. all or part of a gene or mRNA, arestriction fragment of a plasmid or genomic DNA, among others.

As is outlined more fully below, probes (including primers) are made tohybridize to target sequences to determine the presence or absence ofthe target sequence in a sample. Generally speaking, this term will beunderstood by those skilled in the art.

The target sequence may also be comprised of different target domains,for example, in “sandwich” type assays as outlined below, a first targetdomain of the sample target sequence may hybridize to a capture probe orcapture extender probe and a second target domain may hybridize to aportion of an amplifier probe, a label probe, or a different capture orcapture extender probe, etc. In addition, the target domains may beadjacent (i.e. contiguous) or separated. For example, when ligationtechniques are used, a first primer may hybridize to a first targetdomain and a second primer may hybridize to a second target domain;either the domains are adjacent, or they may be separated by one or morenucleotides, coupled with the use of a polymerase and dNTPs, as is morefully outlined below.

The terms “first” and “second” are not meant to confer an orientation ofthe sequences with respect to the 5′-3′ orientation of the targetsequence. For example, assuming a 5′-3′ orientation of the complementarytarget sequence, the first target domain may be located either 5′ to thesecond domain, or 3′ to the second domain.

In a preferred embodiment, the target analyte is a protein. As will beappreciated by those in the art, there are a large number of possibleproteinaceous target analytes that may be detected using the presentinvention. By “proteins” or grammatical equivalents herein is meantproteins, oligopeptides and peptides, derivatives and analogs, includingproteins containing non-naturally occurring amino acids and amino acidanalogs, and peptidomimetic structures. The side chains may be in eitherthe (R) or the (S) configuration. In a preferred embodiment, the aminoacids are in the (S) or L-configuration. As discussed below, when theprotein is used as a binding ligand, it may be desirable to utilizeprotein analogs to retard degradation by sample contaminants.

Suitable target analytes include carbohydrates, including but notlimited to, markers for breast cancer (CA15-3, CA 549, CA 27.29),mucin-like carcinoma associated antigen (MCA), ovarian cancer (CA125),pancreatic cancer (DE-PAN-2), and colorectal and pancreatic cancer (CA19, CA 50, CA242).

These target analytes may be present in any number of different sampletypes, including, but not limited to, bodily fluids including blood,lymph, saliva, vaginal and anal secretions, urine, feces, perspirationand tears, and solid tissues, including liver, spleen, bone marrow,lung, muscle, brain, etc.

Accordingly, the present invention provides microfluidic devices for thedetection of target analytes comprising a solid substrate. As outlinedbelow, the substrate making up the microfluidic device (generallyreferred to herein as the “device substrate”) may be the same ordifferent from the substrate of the detection array (generally referredto herein as the “array substrate”, defined below). The solid substratecan be made of a wide variety of materials and can be configured in alarge number of ways, as is discussed herein and will be apparent to oneof skill in the art. In addition, a single device may comprise more thanone substrate; for example, there may be a “sample treatment” cassettethat interfaces with a separate “detection” cassette; a raw sample isadded to the sample treatment cassette and is manipulated to prepare thesample for detection, which is removed from the sample treatmentcassette and added to the detection cassette. There may be an additionalfunctional cassette into which the device fits; for example, a heatingelement which is placed in contact with the sample cassette to effectreactions such as PCR. In some cases, a portion of the substrate may beremovable; for example, the sample cassette may have a detachabledetection cassette, such that the entire sample cassette is notcontacted with the detection apparatus. See for example U.S. Pat. No.5,603,351 and PCT US96/17116, hereby incorporated by reference.

The composition of the device substrate will depend on a variety offactors, including the techniques used to create the device, the use ofthe device, the composition of the sample, the analyte to be detected,the size of the wells and microchannels, the presence or absence ofelectronic components, etc. Generally, the devices of the inventionshould be easily sterilizable, exhibit low fluorescence and non-specificbinding, be biocompatible and resist temperature changes.

In a preferred embodiment, the microfluidic solid substrate can be madefrom a wide variety of materials, including, but not limited to, siliconsuch as silicon wafers, silicon dioxide, silicon nitride, glass andfused silica, gallium arsenide, indium phosphide, aluminum, ceramics,polyimide, quartz, plastics, resins and polymers includingpolymethylmethacrylate, acrylics, polyethylene, polyethyleneterepthalate, polycarbonate, polystyrene and other styrene copolymers,polypropylene, polytetrafluoroethylene, superalloys, zircaloy, steel,gold, silver, copper, tungsten, molybdeumn, tantalum, KOVAR, KEVLAR,KAPTON, MYLAR, brass, sapphire, etc. High quality glasses such as highmelting borosilicate or fused silicas may be preferred for their UVtransmission properties when any of the sample manipulation stepsrequire light based technologies. In addition, as outlined herein,portions of the internal surfaces of the device may be coated with avariety of coatings as needed, to reduce non-specific binding, to allowthe attachment of binding ligands, etc.

The devices of the invention can be made in a variety of ways, as willbe appreciated by those in the art. See for example WO96/39260, directedto the formation of fluid-tight electrical conduits; U.S. Pat. No.5,747,169, directed to sealing; and EP 0637996 B1; EP 0637998 B1;WO96/39260; WO97/16835; WO98/13683; WO97/16561; WO97/43629; WO96/39252;WO96/15576; WO96/15450; WO97/37755; and WO97/27324; and U.S. Pat. Nos.5,304,487; 5,071,531; 5,061,336; 5,747,169; 5,296,375; 5,110,745;5,587,128; 5,498,392; 5,643,738; 5,750,015; 5,726,026; 5,35,358;5,126,022; 5,770,029; 5,631,337; 5,569,364; 5,135,627; 5,632,876;5,593,838; 5,585,069; 5,637,469; 5,486,335; 5,755,942; 5,681,484; and5,603,351, all of which are hereby incorporated by reference. Suitablefabrication techniques again will depend on the choice of substrate, butpreferred methods include, but are not limited to, a variety ofmicromachining and microfabrication techniques, including filmdeposition processes such as spin coating, chemical vapor deposition,laser fabrication, photolithographic and other etching techniques usingeither wet chemical processes or plasma processes, embossing, injectionmolding, and bonding techniques (see U.S. Pat. No. 5,747,169, herebyincorporated by reference). In addition, there are printing techniquesfor the creation of desired fluid guiding pathways; that is, patterns ofprinted material can permit directional fluid transport. Thus, thebuild-up of “ink” can serve to define a flow channel. In addition, theuse of different “inks” or “pastes” can allow different portions of thepathways having different flow properties. For example, materials can beused to change solute/solvent RF values (the ratio of the distance movedby a particular solute to that moved by a solvent front). For example,printed fluid guiding pathways can be manufactured with a printed layeror layers comprised of two different materials, providing differentrates of fluid transport. Multi-material fluid guiding pathways can beused when it is desirable to modify retention times of reagents in fluidguiding pathways. Furthermore, printed fluid guiding pathways can alsoprovide regions containing reagent substances, by including the reagentsin the “inks” or by a subsequent printing step. See for example U.S.Pat. No. 5,795,453, herein incorporated by reference in its entirety.

In a preferred embodiment, the solid substrate is configured forhandling a single sample that may contain a plurality of targetanalytes. That is, a single sample is added to the device and the samplemay either be aliquoted for parallel processing for detection of theanalytes or the sample may be processed serially, with individualtargets being detected in a serial fashion.

In a preferred embodiment, the solid substrate is configured forhandling multiple samples, each of which may contain one or more targetanalytes. In general, in this embodiment, each sample is handledindividually; that is, the manipulations and analyses are done inparallel, with preferably no contact or contamination between them.Alternatively, there may be some steps in common; for example, it may bedesirable to process different samples separately but detect all of thetarget analytes on a single detection array, as described below.

In addition, it should be understood that while most of the discussionherein is directed to the use of planar substrates with microchannelsand wells, other geometries can be used as well. For example, two ormore planar substrates can be stacked to produce a three dimensionaldevice, that can contain microchannels flowing within one plane orbetween planes; similarly, wells may span two or more substrates toallow for larger sample volumes. Thus for example, both sides of asubstrate can be etched to contain microchannels; see for example U.S.Pat. Nos. 5,603,351 and 5,681,484, both of which are hereby incorporatedby reference.

Thus, the devices of the invention include at least one microchannel orflow channel that allows the flow of sample from the sample inlet portto the other components or modules of the system. The collection ofmicrochannels and wells is sometimes referred to in the art as a“mesoscale flow system”. As will be appreciated by those in the art, theflow channels may be configured in a wide variety of ways, depending onthe use of the channel. For example, a single flow channel starting atthe sample inlet port may be separated into a variety of smallerchannels, such that the original sample is divided into discretesubsamples for parallel processing or analysis. Alternatively, severalflow channels from different modules, for example the sample inlet portand a reagent storage module may feed together into a mixing chamber ora reaction chamber. As will be appreciated by those in the art, thereare a large number of possible configurations; what is important is thatthe flow channels allow the movement of sample and reagents from onepart of the device to another. For example, the path lengths of the flowchannels may be altered as needed; for example, when mixing and timedreactions are required, longer and sometimes tortuous flow channels canbe used; similarly, longer lengths for separation purposes may also bedesirable. Alternatively, the size of a channel may be changed toincrease or reduce the flow rate of the sample. For example, the size ofa channel may be increased in order to reduce sample flow rate.

In general, the microfluidic devices of the invention are generallyreferred to as “mesoscale” devices. The devices herein are typicallydesigned on a scale suitable to analyze microvolumes, although in someembodiments large samples (e.g. cc's of sample) may be reduced in thedevice to a small volume for subsequent analysis. That is, “mesoscale”as used herein refers to chambers and microchannels that havecross-sectional dimensions on the order of 0.1 μm to 500 μm. Themesoscale flow channels and wells have preferred depths on the order of0.1 μm to 100 μm, typically 2-50 μm. The channels have preferred widthson the order of 2.0 to 500 μm, more preferably 3-100 μm. For manyapplications, channels of 5-50 μm are useful. However, for manyapplications, larger dimensions on the scale of millimeters may be used.Similarly, chambers (sometimes also referred to herein as “wells”) inthe substrates often will have larger dimensions, on the scale of a fewmillimeters.

In addition to the flow channel system, the devices of the invention areconfigured to include one or more of a variety of components, hereinreferred to as “modules”, that will be present on any given devicedepending on its use. These modules include, but are not limited to:sample inlet ports; sample introduction or collection modules; cellhandling modules (for example, for cell lysis, cell removal, cellconcentration, cell separation or capture, cell fusion, cell growth,etc.); separation modules, for example, for electrophoresis, gelfiltration, sedimentation, etc.); reaction modules for chemical orbiological alteration of the sample, including amplification of thetarget analyte (for example, when the target analyte is nucleic acid,amplification techniques are useful, including, but not limited topolymerase chain reaction (PCR), ligase chain reaction (LCR), stranddisplacement amplification (SDA), and nucleic acid sequence basedamplification (NASBA)), chemical, physical or enzymatic cleavage oralteration of the target analyte, or chemical modification of thetarget; fluid pumps; fluid valves; heating modules; storage modules forassay reagents; mixing chambers; and detection modules.

In a preferred embodiment, the devices of the invention include at leastone sample inlet port for the introduction of the sample to the device.This may be part of or separate from a sample introduction or collectionmodule; that is, the sample may be directly fed in from the sample inletport to a separation chamber, or it may be pretreated in a samplecollection well\ or chamber.

By port is meant a point of entry or exit, for example from a channel orwell, that regulates flow of the sample. In one embodiment, the port issealable, that is forms a seal such that the sample will not flow fromsealed reservoir. The port may be a physical barrier to flow, such as astopper or diaphragm. Alternatively, the port or barrier to flow isregulated by flow pressure, electric current and the like.

As one of ordinary skill in the art appreciates, ports may not benecessary at all points of entry or exit between the various wells andchannels. However, when necessary, ports may be included at any entry orexit points. For example, in one embodiment, a sample handling wellcomprises a well inlet port and optionally a well outlet port.Similarly, a detection module comprises an inlet port and an outletport.

In a preferred embodiment, the devices of the invention include a samplecollection module, which can be used to concentrate or enrich the sampleif required; for example, see U.S. Pat. No. 5,770,029, including thediscussion of enrichment channels and enrichment means.

In a preferred embodiment, the devices of the invention include a cellhandling module. This is of particular use when the sample comprisescells that either contain the target analyte or that must be removed inorder to detect the target analyte. Thus, for example, the detection ofparticular antibodies in blood can require the removal of the bloodcells for efficient analysis, or the cells must be lysed prior todetection. In this context, “cells” include viral particles that mayrequire treatment prior to analysis, such as the release of nucleic acidfrom a viral particle prior to detection of target sequences. Inaddition, cell handling modules may also utilize a downstream means fordetermining the presence or absence of cells. Suitable cell handlingmodules include, but are not limited to, cell lysis modules, cellremoval modules, cell concentration modules, and cell separation orcapture modules. In addition, as for all the modules of the invention,the cell handling module is in fluid communication via a flow channelwith at least one other module of the invention.

In a preferred embodiment, the cell handling module includes a celllysis module. As is known in the art, cells may be lysed in a variety ofways, depending on the cell type. In one embodiment, as described in EP0 637 998 B1 and U.S. Pat. No. 5,635,358, hereby incorporated byreference, the cell lysis module may comprise cell membrane piercingprotrusions that extend from a surface of the cell handling module. Asfluid is forced through the device, the cells are ruptured. Similarly,this may be accomplished using sharp edged particles trapped within thecell handling region. Alternatively, the cell lysis module can comprisea region of restricted cross-sectional dimension, which results in celllysis upon pressure.

In a preferred embodiment, the cell lysis module comprises a cell lysingagent, such as detergents, NaOH, enzymes, proteinase K, guanidinium HCL,etc. In some embodiments, for example for blood cells, a simple dilutionwith water or buffer can result in hypotonic lysis. The lysis agent maybe solution form, stored within the cell lysis module or in a storagemodule and pumped into the lysis module. Alternatively, the lysis agentmay be in solid form, that is taken up in solution upon introduction ofthe sample. Temperature or mixing may also be applied.

The cell lysis module may also include, either internally or externally,a filtering module for the removal of cellular debris as needed. Thisfilter may be microfabricated between the cell lysis module and thesubsequent module to enable the removal of the lysed cell membrane andother cellular debris components; examples of suitable filters are shownin EP 0 637 998 B1, incorporated by reference.

In a preferred embodiment, the cell handling module includes a cellseparation or capture module. This embodiment utilizes a cell captureregion comprising binding sites capable of reversibly binding a cellsurface molecule to enable the selective isolation (or removal) of aparticular type of cell from the sample population. These bindingmoieties may be immobilized either on the surface of the module or on aparticle trapped within the module (i.e. a bead) by physical absorptionor by covalent attachment. Suitable binding moieties will depend on thecell type to be isolated or removed, and generally includes antibodiesand other binding ligands, such as ligands for cell surface receptors,etc. Thus, a particular cell type may be removed from a sample prior tofurther handling, or the assay is designed to specifically bind thedesired cell type, wash away the non-desirable cell types, followed byeither release of the bound cells by the addition of reagents orsolvents, physical removal (i.e. higher flow rates or pressures), oreven in situ lysis.

Alternatively, a cellular “sieve” can be used to separate cells on thebasis of size or shape. This can be done in a variety of ways, includingprotrusions from the surface that allow size exclusion, a series ofnarrowing channels, or a diafiltration type setup.

In a preferred embodiment, the cell handling module includes a cellremoval module. This may be used when the sample contains cells that arenot required in the assay. Generally, cell removal will be done on thebasis of size exclusion as for “sieving”, above, with channels exitingthe cell handling module that are too small for the cells; filtrationand centrifugation may also be done.

In a preferred embodiment, the cell handling module includes a cellconcentration module. As will be appreciated by those in the art, thisis done using “sieving” methods, for example to concentrate the cellsfrom a large volume of sample fluid prior to lysis, or centrifugation.

In a preferred embodiment, the devices of the invention include aseparation module. Separation in this context means that at least onecomponent of the sample is separated from other components of thesample. This can comprise the separation or isolation of the targetanalyte, or the removal of contaminants that interfere with the analysisof the target analyte, depending on the assay.

In a preferred embodiment, the separation module includeschromatographic-type separation media such as absorptive phasematerials, including, but not limited to reverse phase materials (C₈ orC₁₈ coated particles, etc.), ion-exchange materials, affinitychromatography materials such as binding ligands, etc. See U.S. Pat. No.5,770,029.

In a preferred embodiment, the separation module utilizes bindingligands, as is generally outlined herein for cell separation or analytedetection. In this embodiment, binding ligands are immobilized (again,either by physical absorption or covalent attachment, described below)within the separation module (again, either on the internal surface ofthe module, on a particle such as a bead, filament or capillary trappedwithin the module, for example through the use of a frit). Suitablebinding moieties will depend on the sample component to be isolated orremoved. By “binding ligand” or grammatical equivalents herein is meanta compound that is used to bind a component of the sample, either acontaminant (for removal) or the target analyte (for enrichment). Insome embodiments, as outlined below, the binding ligand is used to probefor the presence of the target analyte, and that will bind to theanalyte.

As will be appreciated by those in the art, the composition of thebinding ligand will depend on the sample component to be separated.Binding ligands for a wide variety of analytes are known or can bereadily found using known techniques. For example, when the component isa protein, the binding ligands include proteins (particularly includingantibodies or fragments thereof (FAbs, etc.)) or small molecules. Whenthe sample component is a metal ion, the binding ligand generallycomprises traditional metal ion ligands or chelators. Preferred bindingligand proteins include peptides. For example, when the component is anenzyme, suitable binding ligands include substrates and inhibitors.Antigen-antibody pairs, receptor-ligands, and carbohydrates and theirbinding partners are also suitable component-binding ligand pairs. Thebinding ligand may be nucleic acid, when nucleic acid binding proteinsare the targets; alternatively, as is generally described in U.S. Pat.Nos. 5,270,163, 5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867,5,705,337, and related patents, hereby incorporated by reference,nucleic acid “aptomers” can be developed for binding to virtually anytarget analyte. Similarly, there is a wide body of literature relatingto the development of binding partners based on combinatorial chemistrymethods. In this embodiment, when the binding ligand is a nucleic acid,preferred compositions and techniques are outlined in PCT US97/20014,hereby incorporated by reference.

In a preferred embodiment, the binding of the sample component to thebinding ligand is specific, and the binding ligand is part of a bindingpair. By “specifically bind” herein is meant that the ligand binds thecomponent, for example the target analyte, with specificity sufficientto differentiate between the analyte and other components orcontaminants of the test sample. The binding should be sufficient toremain bound under the conditions of the separation step or assay,including wash steps to remove non-specific binding. In someembodiments, for example in the detection of certain biomolecules, thedisassociation constants of the analyte to the binding ligand will beless than about 10⁻⁴-10⁻⁶ M⁻¹, with less than about 10⁻⁵ to 10⁻⁹ M⁻¹being preferred and less than about 10⁻⁷-10⁻⁹ M⁻¹ being particularlypreferred.

As will be appreciated by those in the art, the composition of thebinding ligand will depend on the composition of the target analyte.Binding ligands to a wide variety of analytes are known or can bereadily found using known techniques. For example, when the analyte is asingle-stranded nucleic acid, the binding ligand is generally asubstantially complementary nucleic acid. Similarly the analyte may be anucleic acid binding protein and the capture binding ligand is either asingle-stranded or double-stranded nucleic acid; alternatively, thebinding ligand may be a nucleic acid binding protein when the analyte isa single or double-stranded nucleic acid. When the analyte is a protein,the binding ligands include proteins or small molecules. Preferredbinding ligand proteins include peptides. For example, when the analyteis an enzyme, suitable binding ligands include substrates, inhibitors,and other proteins that bind the enzyme, i.e. components of amulti-enzyme (or protein) complex. As will be appreciated by those inthe art, any two molecules that will associate, preferably specifically,may be used, either as the analyte or the binding ligand. Suitableanalyte/binding ligand pairs include, but are not limited to,antibodies/antigens, receptors/ligand, proteins/nucleic acids; nucleicacids/nucleic acids, enzymes/substrates and/or inhibitors, carbohydrates(including glycoproteins and glycolipids)/lectins, carbohydrates andother binding partners, proteins/proteins; and protein/small molecules.These may be wild-type or derivative sequences. In a preferredembodiment, the binding ligands are portions (particularly theextracellular portions) of cell surface receptors that are known tomultimerize, such as the growth hormone receptor, glucose transporters(particularly GLUT4 receptor), transferrin receptor, epidermal growthfactor receptor, low density lipoprotein receptor, high densitylipoprotein receptor, leptin receptor, interleukin receptors includingIL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12,IL-13, IL-15 and IL-17 receptors, VEGF receptor, PDGF receptor, EPOreceptor, TPO receptor, ciliary neurotrophic factor receptor, prolactinreceptor, and T-cell receptors.

When the sample component bound by the binding ligand is the targetanalyte, it may be released for detection purposes if necessary, usingany number of known techniques, depending on the strength of the bindinginteraction, including changes in pH, salt concentration, temperature,etc. or the addition of competing ligands, etc.

In a preferred embodiment, the separation module includes anelectrophoresis module, as is generally described in U.S. Pat. Nos.5,770,029; 5,126,022; 5,631,337; 5,569,364; 5,750,015, and 5,135,627,all of which are hereby incorporated by reference. In electrophoresis,molecules are primarily separated by different electrophoreticmobilities caused by their different molecular size, shape and/orcharge. Microcapillary tubes have recently been used for use inmicrocapillary gel electrophoresis (high performance capillaryelectrophoresis (HPCE)). One advantage of HPCE is that the heatresulting from the applied electric field is efficiently disippated dueto the high surface area, thus allowing fast separation. Theelectrophoresis module serves to separate sample components by theapplication of an electric field, with the movement of the samplecomponents being due either to their charge or, depending on the surfacechemistry of the microchannel, bulk fluid flow as a result ofelectroosmotic flow (EOF).

As will be appreciated by those in the art, the electrophoresis modulecan take on a variety of forms, and generally comprises anelectrophoretic microchannel and associated electrodes to apply anelectric field to the electrophoretic microchannel. Waste fluid outletsand fluid reservoirs are present as required.

The electrodes comprise pairs of electrodes, either a single pair, or,as described in U.S. Pat. Nos. 5,126,022 and 5,750,015, a plurality ofpairs. Single pairs generally have one electrode at each end of theelectrophoretic pathway. Multiple electrode pairs may be used toprecisely control the movement of sample components, such that thesample components may be continuously subjected to a plurality ofelectric fields either simultaneously or sequentially.

In a preferred embodiment, electrophoretic gel media may also be used.By varying the pore size of the media, employing two or more gel mediaof different porosity, and/or providing a pore size gradient, separationof sample components can be maximized. Gel media for separation based onsize are known, and include, but are not limited to, polyacrylamide andagarose. One preferred electrophoretic separation matrix is described inU.S. Pat. No. 5,135,627, hereby incorporated by reference, thatdescribes the use of “mosaic matrix”, formed by polymerizing adispersion of microdomains (“dispersoids”) and a polymeric matrix. Thisallows enhanced separation of target analytes, particularly nucleicacids. Similarly, U.S. Pat. No. 5,569,364, hereby incorporated byreference, describes separation media for electrophoresis comprisingsubmicron to above-micron sized cross-linked gel particles that find usein microfluidic systems. U.S. Pat. No. 5,631,337, hereby incorporated byreference, describes the use of thermoreversible hydrogels comprisingpolyacrylamide backbones with N-substituents that serve to providehydrogen bonding groups for improved electrophoretic separation. Seealso U.S. Pat. Nos. 5,061,336 and 5,071,531, directed to methods ofcasting gels in capillary tubes.

In a preferred embodiment, the devices of the invention include areaction module. This can include either physical, chemical orbiological alteration of one or more sample components. Alternatively,it may include a reaction module wherein the target analyte alters asecond moiety that can then be detected; for example, if the targetanalyte is an enzyme, the reaction chamber may comprise a substrate thatupon modification by the target analyte, can then be detected. In thisembodiment, the reaction module may contain the necessary reagents, orthey may be stored in a storage module and pumped as outlined herein tothe reaction module as needed.

In a preferred embodiment, the reaction module includes a chamber forthe chemical modification of all or part of the sample. For example,chemical cleavage of sample components (CNBr cleavage of proteins, etc.)or chemical cross-linking can be done. PCT US97/07880, herebyincorporated by reference, lists a large number of possible chemicalreactions that can be done in the devices of the invention, includingamide formation, acylation, alkylation, reductive amination, Mitsunobu,Diels Alder and Mannich reactions, Suzuki and Stille coupling, etc.Similarly, U.S. Pat. Nos. 5,616,464 and 5,767,259 describe a variationof ligation chain reaction (LCR; sometimes also referred to asoligonucleotide ligation amplification or OLA) that utilizes a “chemicalligation” of sorts. In this embodiment, similar to LCR, a pair ofprimers are utilized, wherein the first primer is substantiallycomplementary to a first domain of the target and the second primer issubstantially complementary to an adjacent second domain of the target(although, as for LCR, if a “gap” exists, a polymerase and dNTPs may beadded to “fill in” the gap). Each primer has a portion that acts as a“side chain” that does not bind the target sequence and acts one half ofa stem structure that interacts non-covalently through hydrogen bonding,salt bridges, van der Waal's forces, etc. Preferred embodiments utilizesubstantially complementary nucleic acids as the side chains. Thus, uponhybridization of the primers to the target sequence, the side chains ofthe primers are brought into spatial proximity, and, if the side chainscomprise nucleic acids as well, can also form side chain hybridizationcomplexes. At least one of the side chains of the primers comprises anactivatable cross-linking agent, generally covalently attached to theside chain, that upon activation, results in a chemical cross-link orchemical ligation. The activatable group may comprise any moiety thatwill allow cross-linking of the side chains, and include groupsactivated chemically, photonically and thermally, with photoactivatablegroups being preferred. In some embodiments a single activatable groupon one of the side chains is enough to result in cross-linking viainteraction to a functional group on the other side chain; in alternateembodiments, activatable groups are required on each side chain.

In a preferred embodiment, the reaction module includes a chamber forthe biological alteration of all or part of the sample. For example,enzymatic processes including nucleic acid amplification and othernucleic acid modifications including ligation, cleavage,circularization, supercoiling, methylation, acetylation, sequencing,genotyping; hydrolysis of sample components or the hydrolysis ofsubstrates by a target enzyme, the addition or removal of detectablelabels, the addition or removal of phosphate groups, proteinmodification (acylation, glycosylation, addition of lipids,carbohydrates, etc.), the synthesis/modification of small molecules,etc. See also, U.S. Ser. No. 09/553,093 filed Apr. 20, 1999, which isexpressly incorporated herein by reference.

In a preferred embodiment, the target analyte is a nucleic acid and thebiological reaction chamber allows amplification of the target nucleicacid. Suitable amplification techniques include, both targetamplification and probe amplification, including, but not limited to,polymerase chain reaction (PCR), ligase chain reaction (LCR), stranddisplacement amplification (SDA), self-sustained sequence replication(3SR), QB replicase amplification (QBR), repair chain reaction (RCR),cycling probe technology or reaction (CPT or CPR), Invader™, and nucleicacid sequence based amplification (NASBA). Techniques utilizing thesemethods are well known in the art and are described in more detail inU.S. Ser. Nos. 09/553,993, filed Apr. 20, 2000, Ser. No. 09/556,463,fled Apr. 21, 2000 and 60/244,119, filed Oct. 26, 2000, all of which areexpressly incorporated herein by reference. In this embodiment, thereaction reagents generally comprise at least one enzyme (generallypolymerase), primers, and nucleoside triphosphates as needed.

In a preferred embodiment the microfluidic device comprises a pluralityof reaction modules. In this embodiment, the reaction modules mayperform different functions. That is, for example, one reaction moduleperforms PCR while another performs QBR. Alternatively, each reactionmodule performs the same function. What is important is that thereaction modules are connected to a detection module for analysis asoutlined below.

General techniques for nucleic acid amplification are discussed below.In most cases, double stranded target nucleic acids are denatured torender them single stranded so as to permit hybridization of the primersand other probes of the invention. A preferred embodiment utilizes athermal step, generally by raising the temperature of the reaction toabout 95° C., although pH changes and other techniques such as the useof extra probes or nucleic acid binding proteins may also be used.

A probe nucleic acid (also referred to herein as a primer nucleic acid)is then contacted to the target sequence to form a hybridizationcomplex. By “primer nucleic acid” herein is meant a probe nucleic acidthat will hybridize to some portion, i.e. a domain, of the targetsequence. Probes of the present invention are designed to becomplementary to a target sequence (either the target sequence of thesample or to other probe sequences, as is described below), such thathybridization of the target sequence and the probes of the presentinvention occurs. As outlined below, this complementarity need not beperfect; there may be any number of base pair mismatches which willinterfere with hybridization between the target sequence and the singlestranded nucleic acids of the present invention. However, if the numberof mutations is so great that no hybridization can occur under even theleast stringent of hybridization conditions, the sequence is not acomplementary target sequence. Thus, by “substantially complementary”herein is meant that the probes are sufficiently complementary to thetarget sequences to hybridize under normal reaction conditions.

A variety of hybridization conditions may be used in the presentinvention, including high, moderate and low stringency conditions; seefor example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2dEdition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, etal, hereby incorporated by reference. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology—Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993). Generally, stringentconditions are selected to be about 5-10° C. lower than the thermalmelting point (Tm) for the specific sequence at a defined ionic strengthpH. The Tm is the temperature (under defined ionic strength, pH andnucleic acid concentration) at which 50% of the probes complementary tothe target hybridize to the target sequence at equilibrium (as thetarget sequences are present in excess, at Tm, 50% of the probes areoccupied at equilibrium). Stringent conditions will be those in whichthe salt concentration is less than about 1.0 sodium ion, typicallyabout 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes(e.g. 10 to 50 nucleotides) and at least about 60° C. for long probes(e.g. greater than 50 nucleotides). Stringent conditions may also beachieved with the addition of destabilizing agents such as formamide.The hybridization conditions may also vary when a non-ionic backbone,i.e. PNA is used, as is known in the art. In addition, cross-linkingagents may be added after target binding to cross-link, i.e. covalentlyattach, the two strands of the hybridization complex.

Thus, the assays are generally run under stringency conditions whichallows formation of the hybridization complex only in the presence oftarget. Stringency can be controlled by altering a step parameter thatis a thermodynamic variable, including, but not limited to, temperature,formamide concentration, salt concentration, chaotropic saltconcentration pH, organic solvent concentration, etc.

These parameters may also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus it may be desirableto perform certain steps at higher stringency conditions to reducenon-specific binding.

The size of the primer nucleic acid may vary, as will be appreciated bythose in the art, in general varying from 5 to 500 nucleotides inlength, with primers of between 10 and 100 being preferred, between 15and 50 being particularly preferred, and from 10 to 35 being especiallypreferred, depending on the use and amplification technique.

In addition, the different amplification techniques may have furtherrequirements of the primers, as is more fully described below.

Once the hybridization complex between the primer and the targetsequence has been formed, an enzyme, sometimes termed an “amplificationenzyme”, is used to modify the primer. As for all the methods outlinedherein, the enzymes may be added at any point during the assay, eitherprior to, during, or after the addition of the primers. Theidentification of the enzyme will depend on the amplification techniqueused, as is more fully outlined below. Similarly, the modification willdepend on the amplification technique, as outlined below, althoughgenerally the first step of all the reactions herein is an extension ofthe primer, that is, nucleotides are added to the primer to extend itslength.

Once the enzyme has modified the primer to form a modified primer, thehybridization complex is disassociated. Generally, the amplificationsteps are repeated for a period of time to allow a number of cycles,depending on the number of copies of the original target sequence andthe sensitivity of detection, with cycles ranging from 1 to thousands,with from 10 to 100 cycles being preferred and from 20 to 50 cyclesbeing especially preferred.

After a suitable time or amplification, the modified primer is moved toa detection module and incorporated into an assay complex, as is morefully outlined below. The assay complex is attached to a microsphere onan array substrate and then detected, as is described below.

In a preferred embodiment, the amplification is target amplification.Target amplification involves the amplification (replication) of thetarget sequence to be detected, such that the number of copies of thetarget sequence is increased. Suitable target amplification techniquesinclude, but are not limited to, the polymerase chain reaction (PCR),strand displacement amplification (SDA), and nucleic acid sequence basedamplification (NASBA).

In a preferred embodiment, the target amplification technique is PCR.The polymerase chain reaction (PCR) is widely used and described, andinvolve the use of primer extension combined with thermal cycling toamplify a target sequence; see U.S. Pat. Nos. 4,683,195 and 4,683,202,and PCR Essential Data, J. W. Wiley & sons, Ed. C. R. Newton, 1995, allof which are incorporated by reference. In addition, there are a numberof variations of PCR which also find use in the invention, including“quantitative competitive PCR” or “QC-PCR”, “arbitrarily primed PCR” or“AP-PCR”, “immuno-PCR”, “Alu-PCR”, “PCR single strand conformationalpolymorphism” or “PCR-SSCP”, “reverse transcriptase PCR” or “RT-PCR”,“biotin capture PCR”, “vectorette PCR”. “panhandle PCR”, and “PCR selectcDNA subtration”, among others.

In general, PCR may be briefly described as follows. A double strandedtarget nucleic acid is denatured, generally by raising the temperature,and then cooled in the presence of an excess of a PCR primer, which thenhybridizes to the first target strand. A DNA polymerase then acts toextend the primer, resulting in the synthesis of a new strand forming ahybridization complex. The sample is then heated again, to disassociatethe hybridization complex, and the process is repeated. By using asecond PCR primer for the complementary target strand, rapid andexponential amplification occurs. Thus PCR steps are denaturation,annealing and extension. The particulars of PCR are well known, andinclude the use of a thermostable polymerase such as Taq I polymeraseand thermal cycling.

Accordingly, the PCR reaction requires at least one PCR primer and apolymerase. Mesoscale PCR devices are described in U.S. Pat. Nos.5,498,392 and 5,587,128, and WO 97/16561, incorporated by reference.

In a preferred embodiment, the target amplification technique is SDA.Strand displacement amplification (SDA) is generally described in Walkeret al., in Molecular Methods for Virus Detection, Academic Press, Inc.,1995, and U.S. Pat. Nos. 5,455,166 and 5,130,238, all of which arehereby expressly incorporated by reference in their entirety.

In general, SDA may be described as follows. A single stranded targetnucleic acid, usually a DNA target sequence, is contacted with an SDAprimer. An “SDA primer” generally has a length of 25-100 nucleotides,with SDA primers of approximately 35 nucleotides being preferred. An SDAprimer is substantially complementary to a region at the 3′ end of thetarget sequence, and the primer has a sequence at its 5′ end (outside ofthe region that is complementary to the target) that is a recognitionsequence for a restriction endonuclease, sometimes referred to herein asa “nicking enzyme” or a “nicking endonuclease”, as outlined below. TheSDA primer then hybridizes to the target sequence. The SDA reactionmixture also contains a polymerase (an “SDA polymerase”, as outlinedbelow) and a mixture of all four deoxynucleoside-triphosphates (alsocalled deoxynucleotides or dNTPs, i.e. dATP, dTTP, dCTP and dGTP), atleast one species of which is a substituted or modified dNTP; thus, theSDA primer is modified, i.e. extended, to form a modified primer,sometimes referred to herein as a “newly synthesized strand”. Thesubstituted dNTP is modified such that it will inhibit cleavage in thestrand containing the substituted dNTP but will not inhibit cleavage onthe other strand. Examples of suitable substituted dNTPs include, butare not limited, 2′deoxyadenosine 5′-O-(1-thiotriphosphate),5-methyldeoxycytidine 5′-triphosphate, 2′-deoxyuridine 5′-triphosphate,and 7-deaza-2′-deoxyguanosine 5′-triphosphate. In addition, thesubstitution of the dNTP may occur after incorporation into a newlysynthesized strand; for example, a methylase may be used to add methylgroups to the synthesized strand. In addition, if all the nucleotidesare substituted, the polymerase may have 5′→3′ exonuclease activity.However, if less than all the nucleotides are substituted, thepolymerase preferably lacks 5→3′ exonuclease activity.

As will be appreciated by those in the art, the recognitionsite/endonuclease pair can be any of a wide variety of knowncombinations. The endonuclease is chosen to cleave a strand either atthe recognition site, or either 3′ or 5′ to it, without cleaving thecomplementary sequence, either because the enzyme only cleaves onestrand or because of the incorporation of the substituted nucleotides.Suitable recognition site/endonuclease pairs are well known in the art;suitable endonucleases include, but are not limited to, HincII, HindIII,AvaI, Fnu4HI, TthIIII, NclI, BstXI, BamI, etc. A chart depictingsuitable enzymes, and their corresponding recognition sites and themodified dNTP to use is found in U.S. Pat. No. 5,455,166, herebyexpressly incorporated by reference.

Once nicked, a polymerase (an “SDA polymerase”) is used to extend thenewly nicked strand, 5′→3′, thereby creating another newly synthesizedstrand. The polymerase chosen should be able to initiate 5′→3′polymerization at a nick site, should also displace the polymerizedstrand downstream from the nick, and should lack 5′→3′ exonucleaseactivity (this may be additionally accomplished by the addition of ablocking agent). Thus, suitable polymerases in SDA include, but are notlimited to, the Klenow fragment of DNA polymerase I, SEQUENASE 1.0 andSEQUENASE 2.0 (U.S. Biochemical), T5 DNA polymerase and Phi29 DNApolymerase.

Accordingly, the SDA reaction requires, in no particular order, an SDAprimer, an SDA polymerase, a nicking endonuclease, and dNTPs, at leastone species of which is modified.

In general, SDA does not require thermocycling. The temperature of thereaction is generally set to be high enough to prevent non-specifichybridization but low enough to allow specific hybridization; this isgenerally from about 37° C. to about 42° C., depending on the enzymes.

In a preferred embodiment, as for most of the amplification techniquesdescribed herein, a second amplification reaction can be done using thecomplementary target sequence, resulting in a substantial increase inamplification during a set period of time. That is, a second primernucleic acid is hybridized to a second target sequence, that issubstantially complementary to the first target sequence, to form asecond hybridization complex. The addition of the enzyme, followed bydisassociation of the second hybridization complex, results in thegeneration of a number of newly synthesized second strands.

In this way, a number of target molecules are made, and transferred to adetection module, described below. As is more fully outlined below,these reactions (that is, the products of these reactions) can bedetected in a number of ways. In general, either direct or indirectdetection of the target products can be done. “Direct” detection as usedin this context, as for the other amplification strategies outlinedherein, requires the incorporation of a label, either through theincorporation of the label in the amplification primers or by polymeraseincorporation of labeled nucleotides into the growing strand.Alternatively, indirect detection proceeds as a sandwich assay, with thenewly synthesized strands containing few or no labels. Detection thenproceeds via the use of label probes comprising a fluorescent label;these label probes will hybridize either directly to the newlysynthesized strand or to intermediate probes such as amplificationprobes.

In a preferred embodiment, the target amplification technique is nucleicacid sequence based amplification (NASBA). NASBA is generally describedin U.S. Pat. No. 5,409,818 and “Profiting from Gene-based Diagnostics”,CTB International Publishing Inc., N.J., 1996, both of which areexpressly incorporated by reference in their entirety.

In general, NASBA may be described as follows. A single stranded targetnucleic acid, usually an RNA target sequence (sometimes referred toherein as “the first target sequence” or “the first template”), iscontacted with a first NASBA primer. A “NASBA primer” generally has alength of 25-100 nucleotides, with NASBA primers of approximately 50-75nucleotides being preferred. The first NASBA primer is preferably a DNAprimer that has at its 3′ end a sequence that is substantiallycomplementary to the 3′ end of the first template. The first NASBAprimer has an RNA polymerase promoter at its 5′ end. The first NASBAprimer is then hybridized to the first template to form a firsthybridization complex. The NASBA reaction mixture also includes areverse transcriptase enzyme (an “NASBA reverse transcriptase”) and amixture of the four dNTPs, such that the first NASBA primer is modified,i.e. extended, to form a modified first primer, comprising ahybridization complex of RNA (the first template) and DNA (the newlysynthesized strand).

By “reverse transcriptase” or “RNA-directed DNA polymerase” herein ismeant an enzyme capable of synthesizing DNA from a DNA primer and an RNAtemplate. Suitable RNA-directed DNA polymerases include, but are notlimited to, avian myloblastosis virus reverse transcriptase (“AMV RT”)and the Moloney murine leukemia virus RT.

In addition to the components listed above, the NASBA reaction alsoincludes an RNA degrading enzyme, also sometimes referred to herein as aribonuclease, that will hydrolyze RNA of an RNA:DNA hybrid withouthydrolyzing single- or double-stranded RNA or DNA. Suitableribonucleases include, but are not limited to, RNase H from E. coli andcalf thymus.

The ribonuclease degrades the first RNA template in the hybridizationcomplex, resulting in a disassociation of the hybridization complexleaving a first single stranded newly synthesized DNA strand, sometimesreferred to herein as “the second template”.

In addition, the NASBA reaction also includes a second NASBA primer,generally comprising DNA (although as for all the probes herein,including primers, nucleic acid analogs may also be used). This secondNASBA primer has a sequence at its 3′ end that is substantiallycomplementary to the 3′ end of the second template, and also contains anantisense sequence for a functional promoter and the antisense sequenceof a transcription initiation site. Thus, this primer sequence, whenused as a template for synthesis of the third DNA template, containssufficient information to allow specific and efficient binding of an RNApolymerase and initiation of transcription at the desired site.Preferred embodiments utilizes the antisense promoter and transcriptioninitiation site are that of the T7 RNA polymerase, although other RNApolymerase promoters and initiation sites can be used as well, asoutlined below.

The second primer hybridizes to the second template, and a DNApolymerase, also termed a “DNA-directed DNA polymerase”, also present inthe reaction, synthesizes a third template (a second newly synthesizedDNA strand), resulting in second hybridization complex comprising twonewly synthesized DNA strands.

Finally, the inclusion of an RNA polymerase and the required fourribonucleoside triphosphates (ribonucleotides or NTPs) results in thesynthesis of an RNA strand (a third newly synthesized strand that isessentially the same as the first template). The RNA polymerase,sometimes referred to herein as a “DNA-directed RNA polymerase”,recognizes the promoter and specifically initiates RNA synthesis at theinitiation site. In addition, the RNA polymerase preferably synthesizesseveral copies of RNA per DNA duplex. Preferred RNA polymerases include,but are not limited to, T7 RNA polymerase, and other bacteriophage RNApolymerases including those of phage T3, phage φII, Salmonella phagesp6, or Pseudomonase phage gh-1.

Accordingly, the NASBA reaction requires, in no particular order, afirst NASBA primer, a second NASBA primer comprising an antisensesequence of an RNA polymerase promoter, an RNA polymerase thatrecognizes the promoter, a reverse transcriptase, a DNA polymerase, anRNA degrading enzyme, NTPs and dNTPs, in addition to the detectioncomponents outlined below.

These components result in a single starting RNA template generating asingle DNA duplex; however, since this DNA duplex results in thecreation of multiple RNA strands, which can then be used to initiate thereaction again, amplification proceeds rapidly.

As outlined herein, the detection of the newly synthesized strands canproceed in several ways. Direct detection can be done in the detectionmodule when the newly synthesized strands comprise ETM labels, either byincorporation into the primers or by incorporation of modified labellednucleotides into the growing strand. Alternatively, as is more fullyoutlined below, indirect detection of unlabelled strands (which nowserve as “targets” in the detection mode) can occur using a variety ofsandwich assay configurations. As will be appreciated by those in theart, it is preferable to detect DNA strands during NASBA since thepresence of the ribonuclease makes the RNA strands potentially labile.

In a preferred embodiment, the amplification technique is signalamplification. Signal amplification involves the use of limited numberof target molecules as templates to either generate multiple signallingprobes or allow the use of multiple signalling probes. Signalamplification strategies include LCR, CPT, Invader™ technology and theuse of amplification probes in sandwich assays.

In a preferred embodiment, the signal amplification technique is LCR.The method can be run in two different ways; in a first embodiment, onlyone strand of a target sequence is used as a template for ligation;alternatively, both strands may be used. See generally U.S. Pat. Nos.5,185,243 and 5,573,907; EP 0 320 308 B1; EP 0 336 731 B1; EP 0 439 182B1; WO 90/01069; WO 89/12696; and WO 89/09835, and U.S.S.N.s 60/078,102and 60/073,011, all of which are incorporated by reference.

In a preferred embodiment, the single-stranded target sequence comprisesa first target domain and a second target domain, and a first LCR primerand a second LCR primer nucleic acids are added, that are substantiallycomplementary to its respective target domain and thus will hybridize tothe target domains. These target domains may be directly adjacent, i.e.contiguous, or separated by a number of nucleotides. If they arenon-contiguous, nucleotides are added along with means to joinnucleotides, such as a polymerase, that will add the nucleotides to oneof the primers. The two LCR primers are then covalently attached, forexample using a ligase enzyme such as is known in the art. This forms afirst hybridization complex comprising the ligated probe and the targetsequence. This hybridization complex is then denatured (disassociated),and the process is repeated to generate a pool of ligated probes. Inaddition, it may be desirable to have the detection probes, describedbelow, comprise a mismatch at the probe junction site, such that thedetection probe cannot be used as a template for ligation.

In a preferred embodiment, LCR is done for two strands of adouble-stranded target sequence. The target sequence is denatured, andtwo sets of probes are added: one set as outlined above for one strandof the target, and a separate set (i.e. third and fourth primer robenucleic acids) for the other strand of the target. In a preferredembodiment, the first and third probes will hybridize, and the secondand fourth probes will hybridize, such that amplification can occur.That is, when the first and second probes have been attached, theligated probe can now be used as a template, in addition to the secondtarget sequence, for the attachment of the third and fourth probes.Similarly, the ligated third and fourth probes will serve as a templatefor the attachment of the first and second probes, in addition to thefirst target strand. In this way, an exponential, rather than just alinear, amplification can occur.

Again, as outlined above, the detection of the LCR reaction can occurdirectly, in the case where one or both of the primers comprises atleast one label, or indirectly, using sandwich assays, through the useof additional probes; that is, the ligated probes can serve as targetsequences, and detection may utilize amplification probes, captureprobes, capture extender probes, label probes, and label extenderprobes, etc.

Invader™ technology is based on structure-specific polymerases thatcleave nucleic acids in a site-specific manner. Two probes are used: an“invader” probe and a “signalling” probe, that adjacently hybridize to atarget sequence with a non-complementary overlap. The enzyme cleaves atthe overlap due to its recognition of the “tail”, and releases the“tail” with a label. This can then be detected. The Invader™ technologyis described in U.S. Pat. Nos. 5,846,717; 5,614,402; 5,719,028;5,541,311; and 5,843,669, all of which are hereby incorporated byreference.

In a preferred embodiment, the signal amplification technique is CPT.CPT technology is described in a number of patents and patentapplications, including U.S. Pat. Nos. 5,011,769, 5,403,711, 5,660,988,and 4,876,187, and PCT published applications WO 95/05480, WO 95/1416,and WO 95/00667, and U.S. Ser. No. 09/014,304, all of which areexpressly incorporated by reference in their entirety.

Generally, CPT may be described as follows. A CPT primer (also sometimesreferred to herein as a “scissile primer”), comprises two probesequences separated by a scissile linkage. The CPT primer issubstantially complementary to the target sequence and thus willhybridize to it to form a hybridization complex. The scissile linkage iscleaved, without cleaving the target sequence, resulting in the twoprobe sequences being separated. The two probe sequences can thus bemore easily disassociated from the target, and the reaction can berepeated any number of times. The cleaved primer is then detected asoutlined herein.

By “scissile linkage” herein is meant a linkage within the scissileprobe that can be cleaved when the probe is part of a hybridizationcomplex, that is, when a double-stranded complex is formed. It isimportant that the scissile linkage cleave only the scissile probe andnot the sequence to which it is hybridized (i.e. either the targetsequence or a probe sequence), such that the target sequence may bereused in the reaction for amplification of the signal. As used herein,the scissile linkage, is any connecting chemical structure which joinstwo probe sequences and which is capable of being selectively cleavedwithout cleavage of either the probe sequences or the sequence to whichthe scissile probe is hybridized. The scissile linkage may be a singlebond, or a multiple unit sequence. As will be appreciated by those inthe art, a number of possible scissile linkages may be used.

In a preferred embodiment, the scissile linkage comprises RNA. Thissystem, previously described in as outlined above, is based on the factthat certain double-stranded nucleases, particularly ribonucleases, willnick or excise RNA nucleosides from a RNA:DNA hybridization complex. Ofparticular use in this embodiment is RNAseH, Exo III, and reversetranscriptase.

In one embodiment, the entire scissile probe is made of RNA, the nickingis facilitated especially when carried out with a double-strandedribonuclease, such as RNAseH or Exo III. RNA probes made entirely of RNAsequences are particularly useful because first, they can be more easilyproduced enzymatically, and second, they have more cleavage sites whichare accessible to nicking or cleaving by a nicking agent, such as theribonucleases. Thus, scissile probes made entirely of RNA do not rely ona scissile linkage since the scissile linkage is inherent in the probe.

In a preferred embodiment, when the scissile linkage is a nucleic acidsuch as RNA, the methods of the invention may be used to detectmismatches, as is generally described in U.S. Pat. Nos. 5,660,988, andWO 95/14106, hereby expressly incorporated by reference. These mismatchdetection methods are based on the fact that RNAseH may not bind toand/or cleave an RNA:DNA duplex if there are mismatches present in thesequence. Thus, in the NA₁-R-NA₂ embodiments, NA₁ and NA₂ are non-RNAnucleic acids, preferably DNA. Preferably, the mismatch is within theRNA:DNA duplex, but in some embodiments the mismatch is present in anadjacent sequence very close to the desired sequence, close enough toaffect the RNAseH (generally within one or two bases). Thus, in thisembodiment, the nucleic acid scissile linkage is designed such that thesequence of the scissile linkage reflects the particular sequence to bedetected, i.e. the area of the putative mismatch.

In some embodiments of mismatch detection, the rate of generation of thereleased fragments is such that the methods provide, essentially, ayes/no result, whereby the detection of the virtually any releasedfragment indicates the presence of the desired target sequence.Typically, however, when there is only a minimal mismatch (for example,a 1-, 2- or 3-base mismatch, or a 3-base detection), there is somegeneration of cleaved sequences even though the target sequence is notpresent. Thus, the rate of generation of cleaved fragments, and/or thefinal amount of cleaved fragments, is quantified to indicate thepresence or absence of the target. In addition, the use of secondary andtertiary scissile probes may be particularly useful in this embodiment,as this can amplify the differences between a perfect match and amismatch. These methods may be particularly useful in the determinationof homozygotic or heterozygotic states of a patient.

In this embodiment, it is an important feature of the scissile linkagethat its length is determined by the suspected difference between thetarget and the probe. In particular, this means that the scissilelinkage must be of sufficient length to encompass the suspecteddifference, yet short enough the scissile linkage cannot inappropriately“specifically hybridize” to the selected nucleic acid molecule when thesuspected difference is present; such inappropriate hybridization wouldpermit excision and thus cleavage of scissile linkages even though theselected nucleic acid molecule was not fully complementary to thenucleic acid probe. Thus in a preferred embodiment, the scissile linkageis between 3 to 5 nucleotides in length, such that a suspectednucleotide difference from 1 nucleotide to 3 nucleotides is encompassedby the scissile linkage, and 0, 1 or 2 nucleotides are on either side ofthe difference.

Thus, when the scissile linkage is nucleic acid, preferred embodimentsutilize from 1 to about 100 nucleotides, with from about 2 to about 20being preferred and from about 5 to about 10 being particularlypreferred.

CPT may be done enzymatically or chemically. That is, in addition toRNAseH, there are several other cleaving agents which may be useful incleaving RNA (or other nucleic acid) scissile bonds. For example,several chemical nucleases have been reported; see for example Sigman etal., Annu. Rev. Biochem. 1990, 59, 207-236; Sigman et al., Chem. Rev.1993, 93, 2295-2316; Bashkin et al., J. Org. Chem. 1990, 55, 5125-5132;and Sigman et al., Nucleic Acids and Molecular Biology, vol. 3, F.Eckstein and D. M. J. Lilley (Eds), Springer-Verlag, Heidelberg 1989,pp. 13-27; all of which are hereby expressly incorporated by reference.

Specific RNA hydrolysis is also an active area; see for example Chin,Acc. Chem. Res. 1991, 24, 145-152; Breslow et al., Tetrahedron, 1991,47, 2365-2376; Anslyn et al., Angew. Chem. Int. Ed. Engl., 1997, 36,432-450; and references therein, all of which are expressly incorporatedby reference. Reactive phosphate centers are also of interest indeveloping scissile linkages, see Hendry et al., Prog. Inorg. Chem.:Bioinorganic Chem. 1990, 31, 201-258 also expressly incorporated byreference.

Current approaches to site-directed RNA hydrolysis include theconjugation of a reactive moiety capable of cleaving phosphodiesterbonds to a recognition element capable of sequence-specificallyhybridizing to RNA. In most cases, a metal complex is covalentlyattached to a DNA strand which forms a stable heteroduplex. Uponhybridization, a Lewis acid is placed in close proximity to the RNAbackbone to effect hydrolysis; see Magda et al., J. Am. Chem. Soc. 1994,116, 7439; Hall et al., Chem. Biology 1994, 1, 185-190; Bashkin et al.,J. Am. Chem. Soc. 1994, 116, 5981-5982; Hall et al., Nucleic Acids Res.1996, 24, 3522; Magda et al., J. Am. Chem. Soc. 1997, 119, 2293; andMagda et al., J. Am. Chem. Soc. 1997, 119, 6947, all of which areexpressly incorporated by reference.

In a similar fashion, DNA-polyamine conjugates have been demonstrated toinduce site-directed RNA strand scission; see for example, Yoshinari etal., J. Am. Chem. Soc. 1991, 113, 5899-5901; Endo et al., J. Org. Chem.1997, 62, 846; and Barbier et al., J. Am. Chem. Soc. 1992, 114,3511-3515, all of which are expressly incorporated by reference.

In a preferred embodiment, the scissile linkage is not necessarily RNA.For example, chemical cleavage moieties may be used to cleave basicsites in nucleic acids; see Belmont, et al., New J. Chem. 1997, 21,47-54; and references therein, all of which are expressly incorporatedherein by reference. Similarly, photocleavable moieties, for example,using transition metals, may be used; see Moucheron, et al., Inorg.Chem. 1997, 36, 584-592, hereby expressly by reference.

Other approaches rely on chemical moieties or enzymes; see for exampleKeck et al., Biochemistry 1995, 34, 12029-12037; Kirk et al., Chem.Commun. 1998, in press; cleavage of G-U basepairs by metal complexes;see Biochemistry, 1992, 31, 5423-5429; diamine complexes for cleavage ofRNA; Komiyama, et al., J. Org. Chem. 1997, 62, 2155-2160; and Chow etal., Chem. Rev. 1997, 97, 1489-1513, and references therein, all ofwhich are expressly incorporated herein by reference.

The first step of the CPT method requires hybridizing a primary scissileprimer (also called a primary scissile probe) the target. This ispreferably done at a temperature that allows both the binding of thelonger primary probe and disassociation of the shorter cleaved portionsof the primary probe, as will be appreciated by those in the art. Asoutlined herein, this may be done in solution, or either the target orone or more of the scissile probes may be attached to a solid support.For example, it is possible to utilize “anchor probes” on a solidsupport on the array substrate that are substantially complementary to aportion of the target sequence, preferably a sequence that is not thesame sequence to which a scissile probe will bind.

Similarly, as outlined herein, a preferred embodiment has one or more ofthe scissile probes attached to a solid support such as a bead (theseamplification beads are to be distinguished from the detection arraybeads outlined below). In this embodiment, the soluble target diffusesto allow the formation of the hybridization complex between the solubletarget sequence and the support-bound scissile probe. In thisembodiment, it may be desirable to include additional scissile linkagesin the scissile probes to allow the release of two or more probesequences, such that more than one probe sequence per scissile probe maybe detected, as is outlined below, in the interests of maximizing thesignal.

In this embodiment (and in other techniques herein), preferred methodsutilize cutting or shearing techniques to cut the nucleic acid samplecontaining the target sequence into a size that will allow sufficientdiffusion of the target sequence to the surface of a bead. This may beaccomplished by shearing the nucleic acid through mechanical forces orby cleaving the nucleic acid using restriction endonucleases.Alternatively, a fragment containing the target may be generated usingpolymerase, primers and the sample as a template, as in polymerase chainreaction (PCR). In addition, amplification of the target using PCR orLCR or related methods may also be done; this may be particularly usefulwhen the target sequence is present in the sample at extremely low copynumbers. Similarly, numerous techniques are known in the art to increasethe rate of mixing and hybridization including agitation, heating,techniques that increase the overall concentration such asprecipitation, drying, dialysis, centrifugation, electrophoresis,magnetic bead concentration, etc.

In general, the scissile probes are introduced in a molar excess totheir targets (including both the target sequence or other scissileprobes, for example when secondary or tertiary scissile probes areused), with ratios of scissile probe:target of at least about 100:1being preferred, at least about 1000:1 being particularly preferred, andat least about 10,000:1 being especially preferred. In some embodimentsthe excess of probe:target will be much greater. In addition, ratiossuch as these may be used for all the amplification techniques outlinedherein.

Once the hybridization complex between the primary scissile probe andthe target has been formed, the complex is subjected to cleavageconditions. As will be appreciated, this depends on the composition ofthe scissile probe; if it is RNA, RNAseH is introduced. It should benoted that under certain circumstances, such as is generally outlined inWO 95/00666 and WO 95/00667, hereby incorporated by reference, the useof a double-stranded binding agent such as RNAseH may allow the reactionto proceed even at temperatures above the Tm of the primary probe:targethybridization complex. Accordingly, the addition of scissile probe tothe target can be done either first, and then the cleavage agent orcleavage conditions introduced, or the probes may be added in thepresence of the cleavage agent or conditions.

The cleavage conditions result in the separation of the two (or more)probe sequences of the primary scissile probe. As a result, the shorterprobe sequences will no longer remain hybridized to the target sequence,and thus the hybridization complex will disassociate, leaving the targetsequence intact.

The optimal temperature for carrying out the CPT reactions is generallyfrom about 5° C. to about 25° C. below the melting temperatures of theprobe:target hybridization complex. This provides for a rapid rate ofhybridization and high degree of specificity for the target sequence.The Tm of any particular hybridization complex depends on saltconcentration, G-C content, and length of the complex, as is known inthe art and outlined herein.

During the reaction, as for the other amplification techniques herein,it may be necessary to suppress cleavage of the probe, as well as thetarget sequence, by nonspecific nucleases. Such nucleases are generallyremoved from the sample during the isolation of the DNA by heating orextraction procedures. A number of inhibitors of single-strandednucleases such as vanadate, inhibitors it-ACE and RNAsin, a placentalprotein, do not affect the activity of RNAseH. This may not be necessarydepending on the purity of the RNAseH and/or the target sample.

These steps are repeated by allowing the reaction to proceed for aperiod of time. The reaction is usually carried out for about 15 minutesto about 1 hour. Generally, each molecule of the target sequence willturnover between 100 and 1000 times in this period, depending on thelength and sequence of the probe, the specific reaction conditions, andthe cleavage method. For example, for each copy of the target sequencepresent in the test sample 100 to 1000 molecules will be cleaved byRNAseH. Higher levels of amplification can be obtained by allowing thereaction to proceed longer, or using secondary, tertiary, or quaternaryprobes, as is outlined herein.

Upon completion of the reaction, generally determined by time or amountof cleavage, the uncleaved scissile probes must be removed orneutralized prior to detection, such that the uncleaved probe does notbind to a detection probe, causing false positive signals. This may bedone in a variety of ways, as is generally described below.

In a preferred embodiment, the separation is facilitated by the use of asolid support (either an internal surface of the device or beads trappedin the device) containing the primary probe. Thus, when the scissileprobes are attached to the solid support, the flow of the sample pastthis solid support can result in the removal of the uncleaved probes.

In a preferred embodiment, the separation is based on gelelectrophoresis of the reaction products to separate the longeruncleaved probe from the shorter cleaved probe sequences as is known inthe art and described herein.

In a preferred embodiment, the separation is based on strong acidprecipitation. This is useful to separate long (generally greater than50 nucleotides) from smaller fragments (generally about 10 nucleotides).The introduction of a strong acid such as trichloroacetic acid into thesolution (generally from a storage module) causes the longer probe toprecipitate, while the smaller cleaved fragments remain in solution. Theuse of frits or filters can to remove the precipitate, and the cleavedprobe sequences can be quantitated.

In a preferred embodiment, the scissile probe contains both a detectablelabel and an affinity binding ligand or moiety, such that an affinitysupport is used to carry out the separation. In this embodiment, it isimportant that the detectable label used for detection is not on thesame probe sequence that contains the affinity moiety, such that removalof the uncleaved probe, and the cleaved probe containing the affinitymoiety, does not remove all the detectable labels. Suitable affinitymoieties include, but are not limited to, biotin, avidin, streptavidin,lectins, haptens, antibodies, etc. The binding partner of the affinitymoiety is attached to a solid support (again, either an internal surfaceof the device or to beads trapped within the device) and the flow of thesample past this support is used to pull out the uncleaved probes, as isknown in the art. The cleaved probe sequences, which do not contain theaffinity moiety, remain in solution and then can be detected as outlinedbelow.

In a preferred embodiment, similar to the above embodiment, a separationsequence of nucleic acid is included in the scissile probe, which is notcleaved during the reaction. A nucleic acid complementary to theseparation sequence is attached to a solid support and serves as acatcher sequence. Preferably, the separation sequence is added to thescissile probes, and is not recognized by the target sequence, such thata generalized catcher sequence may be utilized in a variety of assays.

In a preferred embodiment, the uncleaved probe is neutralized by theaddition of a substantially complementary neutralization nucleic acid,generally from a storage module. This is particularly useful inembodiments utilizing capture sequences, separation sequences, andone-step systems, as the complement to a probe containing capturesequences forms hybridization complexes that are more stable due to itslength than the cleaved probe sequence:detection probe complex. What isimportant is that the uncleaved probe is not available for binding to adetection probe specific for cleaved sequences. Thus, in one embodiment,this step occurs in the detection module and the neutralization nucleicacid is a detection probe on the surface of the array substrate, at aseparate “address”, such that the signal from the neutralizationhybridization complex does not contribute to the signal of the cleavedfragments. Alternatively, the neutralization nucleic acid may beattached to a solid support; the sample flowed past the neutralizationsurface to quench the reaction, and thus do not enter the detectionmodule.

After removal or neutralization of the uncleaved probe, detectionproceeds via the addition of the cleaved probe sequences to thedetection module, as outlined below.

In a preferred embodiment, no higher order probes are used, anddetection is based on the probe sequence(s) of the primary primer. In apreferred embodiment, at least one, and preferably more, secondaryprobes (also referred to herein as secondary primers) are used. Thesecondary scissile probes may be added to the reaction in several ways.It is important that the secondary scissile probes be prevented fromhybridizing to the uncleaved primary probes, as this results in thegeneration of false positive signal. In a preferred embodiment, theprimary and secondary probes are bound to solid supports. It is onlyupon hybridization of the primary probes with the target, resulting incleavage and release of primary probe sequences from the bead, that thenow diffusible primary probe sequences may bind to the secondary probes.In turn, the primary probe sequences serve as targets for the secondaryscissile probes, resulting in cleavage and release of secondary probesequences. In an alternate embodiment, the complete reaction is done insolution. In this embodiment, the primary probes are added, the reactionis allowed to proceed for some period of time, and the uncleaved primaryscissile probes are removed, as outlined above. The secondary probes arethen added, and the reaction proceeds. The secondary uncleaved probesare then removed, and the cleaved sequences are detected as is generallyoutlined herein. In a preferred embodiment, at least one, and preferablymore, tertiary probes are used. The tertiary scissile probes may beadded to the reaction in several ways. It is important that the tertiaryscissile probes be prevented from hybridizing to the uncleaved secondaryprobes, as this results in the generation of false positive signal.These methods are generally done as outlined above. Similarly,quaternary probes can be used as above.

Thus, CPT requires, again in no particular order, a first CPT primercomprising a first probe sequence, a scissile linkage and a second probesequence; and a cleavage agent.

In this manner, CPT results in the generation of a large amount ofcleaved primers, which then can be detected as outlined below.

In a preferred embodiment, the signal amplification technique is a“sandwich” assay, as is generally described in U.S. Ser. No. 60/073,011and in U.S. Pat. Nos. 5,681,702, 5,597,909, 5,545,730, 5,594,117,5,591,584, 5,571,670, 5,580,731, 5,571,670, 5,591,584, 5,624,802,5,635,352, 5,594,118, 5,359,100, 5,124,246 and 5,681,697, all of whichare hereby incorporated by reference. Although sandwich assays do notresult in the alteration of primers, sandwich assays can be consideredsignal amplification techniques since multiple signals (i.e. labelprobes) are bound to a single target, resulting in the amplification ofthe signal. Sandwich assays are used when the target sequence compriseslittle or no detectable labels; that is, when a secondary probe,comprising the labels, is used to generate the signal.

As discussed herein, it should be noted that the sandwich assays can beused for the detection of primary target sequences (e.g. from a patientsample), or as a method to detect the product of an amplificationreaction as outlined above; thus for example, any of the newlysynthesized strands outlined above, for example using PCR, LCR, NASBA,SDA, etc., may be used as the “target sequence” in a sandwich assay.

Generally, sandwich signal amplification techniques may be described asfollows. The reactions described below can occur either in the reactionmodule, with subsequent transfer to the detection module for detection,or in the detection module with the addition of the required components;for clarity, these are discussed together.

As a preliminary matter, as is more fully described below, captureextender probes may be added to the target sequence for attachment tothe beads in the detection module.

The methods include the addition of an amplifier probe, which ishybridized to the target sequence, either directly, or through the useof one or more label extender probes, which serves to allow “generic”amplifier probes to be made. Preferably, the amplifier probe contains amultiplicity of amplification sequences, although in some embodiments,as described below, the amplifier probe may contain only a singleamplification sequence, or at least two amplification sequences. Theamplifier probe may take on a number of different forms; either abranched conformation, a dendrimer conformation, or a linear “string” ofamplification sequences. Label probes comprising detectable labels thenhybridize to the amplification sequences (or in some cases the labelprobes hybridize directly to the target sequence), and the labels aredetected as is more fully outlined below.

As will be appreciated by those in the art, the systems of the inventionmay take on a large number of different configurations. In general,there are three types of systems that can be used: (1) “non-sandwich”systems (also referred to herein as “direct” detection) in which thetarget sequence itself is labeled (again, either because the primerscomprise labels or due to the incorporation of labeled nucleotides intothe newly synthesized strand); (2) systems in which label probesdirectly bind to the target analytes; and (3) systems in which labelprobes are indirectly bound to the target sequences, for example throughthe use of amplifier probes.

Accordingly, the present invention provides compositions comprising anamplifier probe. By “amplifier probe” or “nucleic acid multimer” or“amplification multimer” or grammatical equivalents herein is meant anucleic acid probe that is used to facilitate signal amplification.Amplifier probes comprise at least a first single-stranded nucleic acidprobe sequence, as defined below, and at least one single-strandednucleic acid amplification sequence, with a multiplicity ofamplification sequences being preferred.

Amplifier probes comprise a first probe sequence that is used, eitherdirectly or indirectly, to hybridize to the target sequence. That is,the amplifier probe itself may have a first probe sequence that issubstantially complementary to the target sequence, or it has a firstprobe sequence that is substantially complementary to a portion of anadditional probe, in this case called a label extender probe, that has afirst portion that is substantially complementary to the targetsequence. In a preferred embodiment, the first probe sequence of theamplifier probe is substantially complementary to the target sequence.

In general, as for all the probes herein, the first probe sequence is ofa length sufficient to give specificity and stability. Thus generally,the probe sequences of the invention that are designed to hybridize toanother nucleic acid (i.e. probe sequences, amplification sequences,portions or domains of larger probes) are at least about 5 nucleosideslong, with at least about 10 being preferred and at least about 15 beingespecially preferred.

In a preferred embodiment, several different amplifier probes are used,each with first probe sequences that will hybridize to a differentportion of the target sequence. That is, there is more than one level ofamplification; the amplifier probe provides an amplification of signaldue to a multiplicity of labelling events, and several differentamplifier probes, each with this multiplicity of labels, for each targetsequence is used. Thus, preferred embodiments utilize at least twodifferent pools of amplifier probes, each pool having a different probesequence for hybridization to different portions of the target sequence;the only real limitation on the number of different amplifier probeswill be the length of the original target sequence. In addition, it isalso possible that the different amplifier probes contain differentamplification sequences, although this is generally not preferred.

In a preferred embodiment, the amplifier probe does not hybridize to thesample target sequence directly, but instead hybridizes to a firstportion of a label extender probe. This is particularly useful to allowthe use of “generic” amplifier probes, that is, amplifier probes thatcan be used with a variety of different targets. This may be desirablesince several of the amplifier probes require special synthesistechniques, for example when branched structures are used. Thus, theaddition of a relatively short probe as a label extender probe ispreferred. Thus, the first probe sequence of the amplifier probe issubstantially complementary to a first portion or domain of a firstlabel extender single-stranded nucleic acid probe. The label extenderprobe also contains a second portion or domain that is substantiallycomplementary to a portion of the target sequence. Both of theseportions are preferably at least about 10 to about 50 nucleotides inlength, with a range of about 15 to about 30 being preferred. The terms“first” and “second” are not meant to confer an orientation of thesequences with respect to the 5′-3′ orientation of the target or probesequences. For example, assuming a 5′-3′ orientation of thecomplementary target sequence, the first portion may be located either5′ to the second portion, or 3′ to the second portion. For convenienceherein, the order of probe sequences are generally described from leftto right.

In a preferred embodiment, more than one label extender probe-amplifierprobe pair may be used. That is, a plurality of label extender probesmay be used, each with a portion that is substantially complementary toa different portion of the target sequence; this can serve as anotherlevel of amplification. Thus, a preferred embodiment utilizes pools ofat least two label extender probes, with the upper limit being set bythe length of the target sequence.

In a preferred embodiment, more than one label extender probe is usedwith a single amplifier probe to reduce non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697, incorporated by referenceherein. In this embodiment, a first portion of the first label extenderprobe hybridizes to a first portion of the target sequence, and thesecond portion of the first label extender probe hybridizes to a firstprobe sequence of the amplifier probe. A first portion of the secondlabel extender probe hybridizes to a second portion of the targetsequence, and the second portion of the second label extender probehybridizes to a second probe sequence of the amplifier probe. These formstructures sometimes referred to as “cruciform” structures orconfigurations, and are generally done to confer stability when largebranched or dendrimeric amplifier probes are used.

In addition, as will be appreciated by those in the art, the labelextender probes may interact with a preamplifier probe, described below,rather than the amplifier probe directly.

Similarly, as outlined above, a preferred embodiment utilizes severaldifferent amplifier probes, each with first probe sequences that willhybridize to a different portion of the label extender probe. Inaddition, as outlined above, it is also possible that the differentamplifier probes contain different amplification sequences, althoughthis is generally not preferred.

In addition to the first probe sequence, the amplifier probe alsocomprises at least one amplification sequence. An “amplificationsequence” or “amplification segment” or grammatical equivalents hereinis meant a sequence that is used, either directly or indirectly, to bindto a first portion of a label probe as is more fully described below(although in some cases the amplification sequence may bind to adetection probe). Preferably, the amplifier probe comprises amultiplicity of amplification sequences, with from about 3 to about 1000being preferred, from about 10 to about 100 being particularlypreferred, and about 50 being especially preferred. In some cases, forexample when linear amplifier probes are used, from 1 to about 20 ispreferred with from about 5 to about 10 being particularly preferred.

The amplification sequences may be linked to each other in a variety ofways, as will be appreciated by those in the art. They may be covalentlylinked directly to each other, or to intervening sequences or chemicalmoieties, through nucleic acid linkages such as phosphodiester bonds,PNA bonds, etc., or through interposed linking agents such amino acid,carbohydrate or polyol bridges, or through other cross-linking agents orbinding partners. The site(s) of linkage may be at the ends of asegment, and/or at one or more internal nucleotides in the strand. In apreferred embodiment, the amplification sequences are attached vianucleic acid linkages.

In a preferred embodiment, branched amplifier probes are used, as aregenerally described in U.S. Pat. No. 5,124,246, hereby incorporated byreference. Branched amplifier probes may take on “fork-like” or“comb-like” conformations. “Fork-like” branched amplifier probesgenerally have three or more oligonucleotide segments emanating from apoint of origin to form a branched structure. The point of origin may beanother nucleotide segment or a multifunctional molecule to which atleast three segments can be covalently or tightly bound. “Comb-like”branched amplifier probes have a linear backbone with a multiplicity ofsidechain oligonucleotides extending from the backbone. In eitherconformation, the pendant segments will normally depend from a modifiednucleotide or other organic moiety having the appropriate functionalgroups for attachment of oligonucleotides. Furthermore, in eitherconformation, a large number of amplification sequences are availablefor binding, either directly or indirectly, to detection probes. Ingeneral, these structures are made as is known in the art, usingmodified multifunctional nucleotides, as is described in U.S. Pat. Nos.5,635,352 and 5,124,246, among others.

In a preferred embodiment, dendrimer amplifier probes are used, as aregenerally described in U.S. Pat. No. 5,175,270, hereby expresslyincorporated by reference. Dendrimeric amplifier probes haveamplification sequences that are attached via hybridization, and thushave portions of double-stranded nucleic acid as a component of theirstructure. The outer surface of the dendrimer amplifier probe has amultiplicity of amplification sequences.

In a preferred embodiment, linear amplifier probes are used, that haveindividual amplification sequences linked end-to-end either directly orwith short intervening sequences to form a polymer. As with the otheramplifier configurations, there may be additional sequences or moietiesbetween the amplification sequences.

In one embodiment, the linear amplifier probe has a single amplificationsequence. However, in a preferred embodiment, linear amplifier probescomprise a multiplicity of amplification sequences.

In addition, the amplifier probe may be totally linear, totallybranched, totally dendrimeric, or any combination thereof.

The amplification sequences of the amplifier probe are used, eitherdirectly or indirectly, to bind to a label probe to allow detection. Ina preferred embodiment, the amplification sequences of the amplifierprobe are substantially complementary to a first portion of a labelprobe. Alternatively, amplifier extender probes are used, that have afirst portion that binds to the amplification sequence and a secondportion that binds to the first portion of the label probe.

In addition, the compositions of the invention may include“preamplifier” molecules, which serves a bridging moiety between thelabel extender molecules and the amplifier probes.

Thus, label probes are either substantially complementary to anamplification sequence or to a portion of the target sequence.

Detection of the amplification reactions of the invention, including thedirect detection of amplification products and indirect detectionutilizing label probes (i.e. sandwich assays), is done by detectingassay complexes comprising labels that are attached to a component ofthe hybridization complex.

In addition, as described in U.S. Pat. No. 5,587,128, the reactionchamber may comprise a composition, either in solution or adhered to thesurface of the reaction chamber, that prevents the inhibition of anamplification reaction by the composition of the well. For example, thewall surfaces may be coated with a silane, for example using asilanization reagent such as dimethylchlorosilane, or coated with asiliconizing reagent such as Aquasil™ or Surfacil™ (Pierce, Rockford,Ill.), which are organosilanes containing a hydrolyzable group. Thishydrolyzable group can hydrolyze in solution to form a silanol that canpolymerize and form a tightly bonded film over the surface of thechamber. The coating may also include a blocking agent that can reactwith the film to further reduce inhibition; suitable blocking agentsinclude amino acid polymers and polymers such as polyvinylpyrrolidone,polyadenylic acid and polymaleimide. Alternatively, for siliconsubstrates, a silicon oxide film may be provided on the walls, or thereaction chamber can be coated with a relatively inert polymer such as apolyvinylchloride. In addition, it may be desirable to add blockingpolynucleotides to occupy any binding sites on the surface of thechamber.

In this and other embodiments, at least one heating and/or coolingmodule may be used, that is either part of the reaction chamber orseparate but can be brought into spatial proximity to the reactionmodule. Suitable heating modules are described in U.S. Pat. Nos.5,498,392 and 5,587,128, and WO 97/16561, incorporated by reference, andmay comprise electrical resistance heaters, pulsed lasers or othersources of electromagnetic energy directed to the reaction chamber. Itshould also be noted that when heating elements are used, it may bedesirable to have the reaction chamber be relatively shallow, tofacilitate heat transfer; see U.S. Pat. No. 5,587,128.

In a preferred embodiment, the biological reaction chamber allowsenzymatic cleavage or alteration of the target analyte. For example,restriction endonucleases may be used to cleave target nucleic acidscomprising target sequences, for example genomic DNA, into smallerfragments to facilitate either amplification or detection.Alternatively, when the target analyte is a protein, it may be cleavedby a protease. Other types of enzymatic hydrolysis may also be done,depending on the composition of the target analyte. In addition, asoutlined herein, the target analyte may comprise an enzyme and thereaction chamber comprises a substrate that is then cleaved to form adetectable product.

In addition, in one embodiment the reaction module includes a chamberfor the physical alteration of all or part of the sample, for examplefor shearing genomic or large nucleic acids, UV crosslinking, etc.

In a preferred embodiment, the devices of the invention include at leastone fluid pump. Pumps generally fall into two categories: “on chip” and“off chip”; that is, the pumps (generally electrode based pumps) can becontained within the device itself, or they can be contained on anapparatus into which the device fits, such that alignment occurs of therequired flow channels to allow pumping of fluids.

In a preferred embodiment, the pumps are contained on the device itself.These pumps are generally electrode based pumps; that is, theapplication of electric fields can be used to move both chargedparticles and bulk solvent, depending on the composition of the sampleand of the device. Suitable on chip pumps include, but are not limitedto, electroosmotic (EO) pumps and electrohydrodynamic (EHD) pumps; theseelectrode based pumps have sometimes been referred to in the art as“electrokinetic (EK) pumps”. All of these pumps rely on configurationsof electrodes placed along a flow channel to result in the pumping ofthe fluids comprising the sample components. As is described in the art,the configurations for each of these electrode based pumps are slightlydifferent; for example, the effectiveness of an EHD pump depends on thespacing between the two electrodes, with the closer together they are,the smaller the voltage required to be applied to effect fluid flow.Alternatively, for EO pumps, the spacing between the electrodes shouldbe larger, with up to one-half the length of the channel in which fluidsare being moved, since the electrode are only involved in applyingforce, and not, as in EHD, in creating charges on which the force willact.

In a preferred embodiment, an electroosmotic pump is used.Electroosmosis (EO) is based on the fact that the surface of manysolids, including quartz, glass and others, become variously charged,negatively or positively, in the presence of ionic materials. Thecharged surfaces will attract oppositely charged counterions in aqueoussolutions. Applying a voltage results in a migration of the counterionsto the oppositely charged electrode, and moves the bulk of the fluid aswell. The volume flow rate is proportional to the current, and thevolume flow generated in the fluid is also proportional to the appliedvoltage. Electroosmostic flow is useful for liquids having someconductivity is and generally not applicable for non-polar solvents. EOpumps are described in U.S. Pat. Nos. 4,908,112 and 5,632,876, PCTUS95/14586 and WO97/43629, incorporated by reference.

In a preferred embodiment, an electrohydrodynamic (EHD) pump is used. InEHD, electrodes in contact with the fluid transfer charge when a voltageis applied. This charge transfer occurs either by transfer or removal ofan electron to or from the fluid, such that liquid flow occurs in thedirection from the charging electrode to the oppositely chargedelectrode. EHD pumps can be used to pump resistive fluids such asnon-polar solvents. EHD pumps are described in U.S. Pat. No. 5,632,876,hereby incorporated by reference.

The electrodes of the pumps preferably have a diameter from about 25microns to about 100 microns, more preferably from about 50 microns toabout 75 microns. Preferably, the electrodes protrude from the top of aflow channel to a depth of from about 5% to about 95% of the depth ofthe channel, with from about 25% to about 50% being preferred. Inaddition, as described in PCT US95/14586, an electrode-based internalpumping system can be integrated into the liquid distribution system ofthe devices of the invention with flow-rate control at multiple pumpsites and with fewer complex electronics if the pumps are operated byapplying pulsed voltages across the electrodes; this gives theadditional advantage of ease of integration into high density systems,reductions in the amount of electrolysis that occurs at electrodes,reductions in thermal convection newar the electrodes, and the abilityto use simpler drivers, and the ability to use both simple and complexpulse wave geometries.

The voltages required to be applied to the electrodes cause fluid flowdepends on the geometry of the electrodes and the properties of thefluids to be moved. The flow rate of the fluids is a function of theamplitude of the applied voltage between electrode, the electrodegeometry and the fluid properties, which can be easily determined foreach fluid. Test voltages used may be up to about 1500 volts, but anoperating voltage of about 40 to 300 volts is desirable. An analogdriver is generally used to vary the voltage applied to the pump from aDC power source. A transfer function for each fluid is determinedexperimentally as that applied voltage that produces the desired flow orfluid pressure to the fluid being moved in the channel. However, ananalog driver is generally required for each pump along the channel andis suitable an operational amplifier.

In a preferred embodiment, a micromechanical pump is used, either on- oroff-chip, as is known in the art.

In a preferred embodiment, an “off-chip” pump is used. For example, thedevices of the invention may fit into an apparatus or appliance that hasa nesting site for holding the device, that can register the ports (i.e.sample inlet ports, fluid inlet ports, and waste outlet ports) andelectrode leads. The apparatus can including pumps that can apply thesample to the device; for example, can force cell-containing samplesinto cell lysis modules containing protrusions, to cause cell lysis uponapplication of sufficient flow pressure. Such pumps are well known inthe art.

In a preferred embodiment, the devices of the invention include at leastone fluid valve that can control the flow of fluid into or out of amodule of the device. A variety of valves are known in the art. Forexample, in one embodiment, the valve may comprise a capillary barrier,as generally described in PCT US97/07880, incorporated by reference. Inthis embodiment, the channel opens into a larger space designed to favorthe formation of an energy minimizing liquid surface such as a meniscusat the opening. Preferably, capillary barriers include a dam that raisesthe vertical height of the channel immediated before the opening into alarger space such a chamber. In addition, as described in U.S. Pat. No.5,858,195, incorporated herein by reference, a type of “virtual valve”can be used.

In a preferred embodiment, the devices of the invention include sealingports, to allow the introduction of fluids, including samples, into anyof the modules of the invention, with subsequent closure of the port toavoid the loss of the sample.

In a preferred embodiment, the devices of the invention include at leastone storage modules for assay reagents. These are connected to othermodules of the system using flow channels and may comprise wells orchambers, or extended flow channels. They may contain any number ofreagents, buffers, salts, etc.

In a preferred embodiment, the devices of the invention include a mixingmodule; again, as for storage modules, these may be extended flowchannels (particularly useful for timed mixing), wells or chambers.Particularly in the case of extended flow channels, there may beprotrusions on the side of the channel to cause mixing.

In a preferred embodiment, the devices of the invention include adetection module. The present invention is directed to methods andcompositions useful in the detection of biological target analytespecies such as nucleic acids and proteins. In general, the detectionmodule is based on work outlined in U.S. Ser. Nos. 09/151,877;09/187,289 and 09/189,543; PCT US98/21193; PCT US99/04473 and PCTUS98/05025, all of which are hereby incorporated by reference in theirentirety.

The detection modules of the present invention comprise an arraysubstrate with a surface comprising discrete sites and a population ofarray microspheres (sometimes referred to herein as beads) distributedon the array surface. The detection module of the microfluidic devicesdescribed herein are based on previous work comprising a bead-basedanalytic chemistry system in which beads, also termed microspheres,carrying different chemical functionalities are distributed on an arraysubstrate comprising a patterned surface of discrete sites that can bindthe individual microspheres. The beads are generally put onto thesubstrate randomly, and thus several different methodologies can be usedto “decode” the arrays. In one embodiment, unique optical signatures areincorporated into the beads, generally fluorescent dyes, that could beused to identify the chemical functionality on any particular bead. Thisallows the synthesis of the candidate agents (i.e. compounds such asnucleic acids and antibodies) to be divorced from their placement on anarray, i.e. the candidate agents may be synthesized on the beads, andthen the beads are randomly distributed on a patterned surface. Sincethe beads are first coded with an optical signature, this means that thearray can later be “decoded”, i.e. after the array is made, acorrelation of the location of an individual site on the array with thebead or candidate agent at that particular site can be made. This meansthat the beads may be randomly distributed on the array, a fast andinexpensive process as compared to either the in situ synthesis orspotting techniques of the prior art. These methods are generallyoutlined in PCT US98/05025, PCT/US99/20914, U.S. Pat. No. 6,023,540 andU.S. Ser. Nos. 09/151,877 and 09/450,829, all of which are expresslyincorporated herein by reference.

However, the drawback to these methods is that for a very high densityarray, the system requires a large number of different opticalsignatures, which may be difficult or time-consuming to utilize.Accordingly, the present invention also provides several improvementsover these methods, generally directed to methods of coding and decodingthe arrays. That is, as will be appreciated by those in the art, theplacement of the bioactive agents is generally random, and thus acoding/decoding system is required to identify the bioactive agent ateach location in the array. This may be done in a variety of ways, as ismore fully outlined below, and generally includes: a) the use a decodingbinding ligand (DBL), generally directly labeled, that binds to eitherthe bioactive agent or to identifier binding ligands (IBLs) attached tothe beads; b) positional decoding, for example by either targeting theplacement of beads (for example by using photoactivatible orphotocleavable moieties to allow the selective addition of beads toparticular locations), or by using either sub-bundles or selectiveloading of the sites, as are more fully outlined below; c) selectivedecoding, wherein only those beads that bind to a target are decoded; ord) combinations of any of these. In some cases, as is more fullyoutlined below, this decoding may occur for all the beads, or only forthose that bind a particular target analyte. Similarly, this may occureither prior to or after addition of a target analyte.

In the detection module of the present invention, “decoding” can useoptical signatures, decoding binding ligands that are added during adecoding step, or a combination of these methods. The decoding bindingligands will bind either to a distinct identifier binding ligand partnerthat is placed on the beads, or to the bioactive agent itself, forexample when the beads comprise single-stranded nucleic acids as thebioactive agents. The decoding binding ligands are either directly orindirectly labeled, and thus decoding occurs by detecting the presenceof the label. By using pools of decoding binding ligands in a sequentialfashion, it is possible to greatly minimize the number of requireddecoding steps.

Once the identity (i.e. the actual agent) and location of eachmicrosphere in the array has been fixed, the detection array is exposedto samples containing the target analytes, although as outlined below,this can be done prior to or during the analysis as well. The componentsof the microfluidic device may be used in the decoding as desired. Thetarget analytes will bind to the bioactive agents as is more fullyoutlined below, and results in a change in the optical signal of aparticular bead, resulting in detection.

Accordingly, the present invention provides detection modules comprisingarrays comprising at least a first substrate with a surface comprising aplurality of assay locations. By “array” herein is meant a plurality ofcandidate agents in an array format; the size of the array will dependon the composition and end use of the array. Arrays containing fromabout 2 different bioactive agents (i.e. different beads) to manymillions can be made, with very large fiber optic arrays being possible.Generally, the array will comprise from two to as many as a billion ormore, depending on the size of the beads and the substrate, as well asthe end use of the array, thus very high density, high density, moderatedensity, low density and very low density arrays may be made. Preferredranges for very high density arrays are from about 10,000,000 to about2,000,000,000, (with all numbers being per square centimeter) with fromabout 100,000,000 to about 1,000,000,000 being preferred. High densityarrays range about 100,000 to about 10,000,000, with from about1,000,000 to about 5,000,000 being particularly preferred. Moderatedensity arrays range from about 10,000 to about 100,000 beingparticularly preferred, and from about 20,000 to about 50,000 beingespecially preferred. Low density arrays are generally less than 10,000,with from about 1,000 to about 5,000 being preferred. Very low densityarrays are less than 1,000, with from about 10 to about 1000 beingpreferred, and from about 100 to about 500 being particularly preferred.In some embodiments, the compositions of the invention may not be inarray format; that is, for some embodiments, compositions comprising asingle bioactive agent may be made as well. In addition, in some arrays,multiple substrates may be used, either of different or identicalcompositions. Thus for example, large arrays may comprise a plurality ofsmaller substrates.

In addition, one advantage of the present compositions is thatparticularly through the use of fiber optic technology, extremely highdensity arrays can be made. Thus for example, because beads of 200 μm orless (with beads of 200 nm possible) can be used, and very small fibersare known, it is possible to have as many as 250,000 or more (in someinstances, 1 million) different fibers and beads in a 1 mm² fiber opticbundle, with densities of greater than 15,000,000 individual beads andfibers (again, in some instances as many as 25-50 million) per 0.5 cm²obtainable.

By “array substrate” or “array solid support” or other grammaticalequivalents herein is meant any material that can be modified to containdiscrete individual sites appropriate for the attachment or associationof beads and is amenable to at least one detection method as outlinedherein. As will be appreciated by those in the art, the number ofpossible array substrates is very large. Possible array substratesinclude, but are not limited to, glass and modified or functionalizedglass, plastics (including acrylics, polystyrene and copolymers ofstyrene and other materials, polypropylene, polyethylene, polybutylene,polyurethanes, TeflonJ, etc.), polysaccharides, nylon or nitrocellulose,resins, silica or silica-based materials including silicon and modifiedsilicon, carbon, metals, inorganic glasses, plastics, optical fiberbundles, and a variety of other polymers. In general, the substratesallow optical detection and do not themselves appreciably fluorescese.The array substrates may be the same as the device substrates, or theymay be different. If different, they may be attached to the device inany number of ways, as will be appreciated by those in the art,including, but not limited to, the use of adhesives, fusing the twomaterials together (for example using heat or organic solvents).

Generally the substrate is flat (planar), although as will beappreciated by those in the art, other configurations of assaysubstrates may be used as well; for example, three dimensionalconfigurations can be used, for example by embedding the beads in aporous block of plastic that allows sample access to the beads and usinga confocal microscope for detection. Similarly, the beads may be placedon the inside surface of a tube, for flow-through sample analysis tominimize sample volume. Preferred assay substrates include optical fiberbundles as discussed below, and flat planar substrates such as glass,polystyrene and other plastics and acrylics.

Accordingly, in a preferred embodiment, the array comprises a fiberoptic bundle. That is, the microfluidic chip and a fiber optic bundle asdescribed in WO/98/40726 and WO/00/016101 are combined to form thedevice of the invention.

In one embodiment, the microfluidic chip and the fiber optic bundle areprepared separately and then combined. To combine the fiber optic bundlewith the microfluidic chip, a hole is opened in the chip that intersectswith a channel in the chip. In a preferred embodiment, the hole isperpendicular to the channel. In addition, it is preferred that the holepenetrate only through the first wall of the channel. Finally, it ispreferred that the diameter of the hole match the diameter of the fiberoptic bundle. When the diameter of the hole does not precisely match thesize of the fiber optic bundle, adapter fittings may be used tofacilitate the connection of the chip with the fiber optic bundle.

Once the opening has been formed in the chip, the fiber optic bundle isinserted into the hole. In a preferred embodiment, the surface for thebundle matches or is aligned with the first wall of the channel.

The bundle is attached to the chip through any of a number of ways as isknown in the art. These include for example, adhesive or press fittings.

In one embodiment, microspheres are distributed on the array or bundleprior to connecting to the microfluidic chip. Alternatively, the beadsare distributed following connection of the bundle to the chip as isoutlined below.

In another preferred embodiment, the substrate comprising discrete sitesis the microfluidic chamber itself. That is, a channel of themicrofluidic device is modified so as to contain wells for distributionof the beads. In one embodiment the wells are made by etching or moldingthe surface of the chamber as described herein. Alternatively, pre-madewells are added, i.e., adfixed, to the floor of the chamber.

The assay substrate comprises an assay surface comprising a plurality ofassay locations, i.e. the location where the assay for the detection ofa target analyte will occur. The assay locations are generallyphysically separated from each other, although other configurations(hydrophobicity/hydrophilicity, etc.) can be used to separate the assaylocations.

In a preferred embodiment, the assay substrate is a slice or a sectionan optical fiber bundle or array, as is generally described in U.S. Ser.Nos. 08/944,850, 08/519,062 and 09/287,573, PCT US98/05025,PCT/US98/21193 and PCT US98/09163, all of which are expresslyincorporated herein by reference. Preferred embodiments utilizepreformed unitary fiber optic arrays. By “preformed unitary fiber opticarray” herein is meant an array of discrete individual fiber opticstrands that are co-axially disposed and joined along their lengths. Thefiber strands are generally individually clad. However, one thing thatdistinguished a preformed unitary array from other fiber optic formatsis that the fibers are not individually physically manipulatable; thatis, one strand generally cannot be physically separated at any pointalong its length from another fiber strand.

In a preferred embodiment, the assay surface comprises a plurality ofdiscrete sites. That is, at least one surface of the substrate ismodified to contain discrete, individual sites for later association ofmicrospheres. These sites may comprise physically altered sites, i.e.physical configurations such as wells or small depressions in thesubstrate that can retain the beads, such that a microsphere can rest inthe well, or the use of other forces (magnetic or compressive), orchemically altered or active sites, such as chemically functionalizedsites, electrostatically altered sites, hydrophobically/hydrophilicallyfunctionalized sites, spots of adhesive, etc.

The sites may be a pattern, i.e. a regular design or configuration, orrandomly distributed. A preferred embodiment utilizes a regular patternof sites such that the sites may be addressed in the X-Y coordinateplane. “Pattern” in this sense includes a repeating unit cell,preferably one that allows a high density of beads on the arraysubstrate. However, it should be noted that these sites may not bediscrete sites. That is, it is possible to use a uniform surface ofadhesive or chemical functionalities, for example, that allows theattachment of beads at any position. That is, the surface of thesubstrate is modified to allow attachment of the microspheres atindividual sites, whether or not those sites are contiguous ornon-contiguous with other sites. Thus, the surface of the substrate maybe modified such that discrete sites are formed that can only have asingle associated bead, or alternatively, the surface of the substrateis modified and beads may go down anywhere, but they end up at discretesites.

In a preferred embodiment, the surface of the array substrate ismodified to contain wells, i.e. depressions in the surface of thesubstrate. This may be done as is generally known in the art using avariety of techniques, including, but not limited to, photolithography,stamping techniques, molding techniques and microetching techniques. Aswill be appreciated by those in the art, the technique used will dependon the composition and shape of the substrate.

In a preferred embodiment, physical alterations are made in a surface ofthe substrate to produce the sites. In a preferred embodiment, forexample when the array substrate is a fiber optic bundle, the surface ofthe substrate is a terminal end of the fiber bundle, as is generallydescribed in PCT US98/05025, PCT/US99/20914, U.S. Pat. No. 6,023,540 andU.S. Ser. Nos. 09/151,877 and 09/450,829, all of which are herebyexpressly incorporated by reference. In this embodiment, wells are madein a terminal or distal end of a fiber optic bundle comprisingindividual fibers. In this embodiment, the cores of the individualfibers are etched, with respect to the cladding, such that small wellsor depressions are formed at one end of the fibers. The required depthof the wells will depend on the size of the beads to be added to thewells.

Generally in this embodiment, the microspheres are non-covalentlyassociated in the wells, although the wells may additionally bechemically functionalized as is generally described below, cross-linkingagents may be used, or a physical barrier may be used, i.e. a film ormembrane over the beads.

In a preferred embodiment, the surface of the array substrate ismodified to contain chemically modified sites, that can be used toattach, either covalently or non-covalently, the microspheres of theinvention to the discrete sites or locations on the substrate.“Chemically modified sites” in this context includes, but is not limitedto, the addition of a pattern of chemical functional groups includingamino groups, carboxy groups, oxo groups and thiol groups, that can beused to covalently attach microspheres, which generally also containcorresponding reactive functional groups; the addition of a pattern ofadhesive that can be used to bind the microspheres (either by priorchemical functionalization for the addition of the adhesive or directaddition of the adhesive); the addition of a pattern of charged groups(similar to the chemical functionalities) for the electrostaticattachment of the microspheres, i.e. when the microspheres comprisecharged groups opposite to the sites; the addition of a pattern ofchemical functional groups that renders the sites differentiallyhydrophobic or hydrophilic, such that the addition of similarlyhydrophobic or hydrophilic microspheres under suitable experimentalconditions will result in association of the microspheres to the siteson the basis of hydroaffinity. For example, the use of hydrophobic siteswith hydrophobic beads, in an aqueous system, drives the association ofthe beads preferentially onto the sites. As outlined above, “pattern” inthis sense includes the use of a uniform treatment of the surface toallow attachment of the beads at discrete sites, as well as treatment ofthe surface resulting in discrete sites. As will be appreciated by thosein the art, this may be accomplished in a variety of ways.

In a preferred embodiment, the substrate is configured to allow mixingof the sample, reagents, microspheres, etc. That is, in a variety ofembodiments, mixing or sample turbulence is desirable. This can beaccomplished in a variety of ways. In a preferred embodiment, thesubstrate comprises raised microstructures such as vertical “posts” orweirs, or other configurations that create sample turbulence, such asedged depressions. These structures may be configured with respect tothe chamber such that the flow of the sample past the array causesmixing or sample turbulence. For example, in one embodiment thedetection surface is “sunken” or “recessed” with respect to the chamber,such that the flow of the sample past the electrode causes mixing. In apreferred embodiment, vertical “posts” or “pins” are included, to createsample turbulence.

These microstructures can be included anywhere within the device,including within chambers or channels, and may be formed from anysubstrate as described herein by known microstructure fabricationtechniques. In one embodiment, these microstructures are formed orcoated from materials different from the substrate to preventundesirable interactions with the beads or sample; for example, in apreferred embodiment, the posts are made of metal.

The compositions of the invention further comprise a population ofmicrospheres. By “population” herein is meant a plurality of beads asoutlined above for arrays. Within the population are separatesubpopulations, which can be a single microsphere or multiple identicalmicrospheres. That is, in some embodiments, as is more fully outlinedbelow, the array may contain only a single bead for each bioactiveagent; preferred embodiments utilize a plurality of beads of each type.

By “microspheres” or “beads” or “particles” or grammatical equivalentsherein is meant small discrete particles. The composition of the beadswill vary, depending on the class of bioactive agent and the method ofsynthesis. Suitable bead compositions include those used in peptide,nucleic acid and organic moiety synthesis, including, but not limitedto, plastics, ceramics, glass, polystyrene, methylstyrene, acrylicpolymers, paramagnetic materials, thoria sol, carbon graphited, titaniumdioxide, latex or cross-linked dextrans such as Sepharose, cellulose,nylon, cross-linked micelles and teflon may all be used. “MicrosphereDetection Guide” from Bangs Laboratories, Fishers Ind. is a helpfulguide.

The beads need not be spherical; irregular particles may be used. Inaddition, the beads may be porous, thus increasing the surface area ofthe bead available for either bioactive agent attachment or tagattachment. The bead sizes range from nanometers, i.e. 100 nm, tomillimeters, i.e. 1 mm, with beads from about 0.2 micron to about 200microns being preferred, and from about 0.5 to about 5 micron beingparticularly preferred, although in some embodiments smaller beads maybe used.

It should be noted that a key component of the invention is the use of asubstrate/bead pairing that allows the association or attachment of thebeads at discrete sites on the surface of the substrate, such that thebeads do not move during the course of the assay.

Each microsphere comprises a bioactive agent, although as will beappreciated by those in the art, there may be some microspheres which donot contain a bioactive agent, depending the on the synthetic methods.By “candidate bioactive agent” or “bioactive agent” or “chemicalfunctionality” or “binding ligand” herein is meant as used hereindescribes any molecule, e.g., protein, oligopeptide, small organicmolecule, coordination complex, polysaccharide, polynucleotide, etc.which can be attached to the microspheres of the invention. It should beunderstood that the compositions of the invention have two primary uses.In a preferred embodiment, as is more fully outlined below, thecompositions are used to detect the presence of a particular targetanalyte; for example, the presence or absence of a particular nucleotidesequence or a particular protein, such as an enzyme, an antibody or anantigen. In an alternate preferred embodiment, the compositions are usedto screen bioactive agents, i.e. drug candidates, for binding to aparticular target analyte.

Bioactive agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons.Bioactive agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The bioactiveagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Bioactive agents are also found amongbiomolecules including peptides, nucleic acids, saccharides, fattyacids, steroids, purines, pyrimidines, derivatives, structural analogsor combinations thereof. Particularly preferred are nucleic acids andproteins.

Bioactive agents can be obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means. Knownpharmacological agents may be subjected to directed or random chemicalmodifications, such as acylation, alkylation, esterification and/oramidification to produce structural analogs.

In a preferred embodiment, the bioactive agents are proteins, as definedabove.

In one preferred embodiment, the bioactive agents are naturallyoccurring proteins or fragments of naturally occurring proteins. Thus,for example, cellular extracts containing proteins, or random ordirected digests of proteinaceous cellular extracts, may be used. Inthis way libraries of procaryotic and eukaryotic proteins may be madefor screening in the systems described herein. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred.

In a preferred embodiment, the bioactive agents are peptides of fromabout 5 to about 30 amino acids, with from about 5 to about 20 aminoacids being preferred, and from about 7 to about 15 being particularlypreferred. The peptides may be digests of naturally occurring proteinsas is outlined above, random peptides, or “biased” random peptides. By“randomized” or grammatical equivalents herein is meant that eachnucleic acid and peptide consists of essentially random nucleotides andamino acids, respectively. Since generally these random peptides (ornucleic acids, discussed below) are chemically synthesized, they mayincorporate any nucleotide or amino acid at any position. The syntheticprocess can be designed to generate randomized proteins or nucleicacids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized bioactive proteinaceous agents.

In a preferred embodiment, a library of bioactive agents are used. Thelibrary should provide a sufficiently structurally diverse population ofbioactive agents to effect a probabilistically sufficient range ofbinding to target analytes. Accordingly, an interaction library must belarge enough so that at least one of its members will have a structurethat gives it affinity for the target analyte. Although it is difficultto gauge the required absolute size of an interaction library, natureprovides a hint with the immune response: a diversity of 10⁷-10⁸different antibodies provides at least one combination with sufficientaffinity to interact with most potential antigens faced by an organism.Published in vitro selection techniques have also shown that a librarysize of 10⁷ to 10⁸ is sufficient to find structures with affinity forthe target. Thus, in a preferred embodiment, at least 10⁶, preferably atleast 10⁷, more preferably at least 10⁸ and most preferably at least 10⁹different bioactive agents are simultaneously analyzed in the subjectmethods. Preferred methods maximize library size and diversity.

In a preferred embodiment, the library is fully randomized, with nosequence preferences or constants at any position. In a preferredembodiment, the library is biased. That is, some positions within thesequence are either held constant, or are selected from a limited numberof possibilities. For example, in a preferred embodiment, thenucleotides or amino acid residues are randomized within a definedclass, for example, of hydrophobic amino acids, hydrophilic residues,sterically biased (either small or large) residues, towards the creationof cysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

In a preferred embodiment, the bioactive agents are nucleic acids asdefined above (generally called “probe nucleic acids” or “candidateprobes” herein). As described above generally for proteins, nucleic acidbioactive agents may be naturally occurring nucleic acids, randomnucleic acids, or “biased” random nucleic acids. For example, digests ofprocaryotic or eukaryotic genomes may be used as is outlined above forproteins.

When the bioactive agents are nucleic acids, they are designed to besubstantially complementary to target sequences. The term “targetsequence” or grammatical equivalents herein means a nucleic acidsequence on a single strand of nucleic acid. The target sequence may bea portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNAincluding mRNA and rRNA, or others. It may be any length, with theunderstanding that longer sequences are more specific. As will beappreciated by those in the art, the complementary target sequence maytake many forms. For example, it may be contained within a largernucleic acid sequence, i.e. all or part of a gene or mRNA, a restrictionfragment of a plasmid or genomic DNA, among others. As is outlined morefully below, probes are made to hybridize to target sequences todetermine the presence or absence of the target sequence in a sample.Generally speaking, this term will be understood by those skilled in theart.

In a preferred embodiment, the bioactive agents are organic chemicalmoieties, a wide variety of which are available in the literature.

In a preferred embodiment, each bead comprises a single type ofbioactive agent, although a plurality of individual bioactive agents arepreferably attached to each bead. Similarly, preferred embodimentsutilize more than one microsphere containing a unique bioactive agent;that is, there is redundancy built into the system by the use ofsubpopulations of microspheres, each microsphere in the subpopulationcontaining the same bioactive agent. The numbers of beads for eachsubpopulation will vary. Those of skill in the art will appreciate thatthe random distribution of the beads on the array substrate willgenerally follow a Poisson distribution, and thus any particularsubpopulation will have the same number or a different number of beadson the array substrate. Similarly, the redundancy of the array will varywith the application for which it is used. Preferred embodiments have atleast two beads of each subpopulation on the array, with from at leastabout three to about fifty being preferred, from about five to abouttwenty being preferred, and from about eight to about ten beingparticularly preferred.

As will be appreciated by those in the art, the bioactive agents mayeither be synthesized directly on the beads, or they may be made andthen attached after synthesis. In a preferred embodiment, linkers areused to attach the bioactive agents to the beads, to allow both goodattachment, sufficient flexibility to allow good interaction with thetarget molecule, and to avoid undesirable binding reactions.

In a preferred embodiment, the bioactive agents are synthesized directlyon the beads. As is known in the art, many classes of chemical compoundsare currently synthesized on solid supports, such as peptides, organicmoieties, and nucleic acids. It is a relatively straightforward matterto adjust the current synthetic techniques to use beads.

In a preferred embodiment, the bioactive agents are synthesized first,and then covalently attached to the beads. As will be appreciated bythose in the art, this will be done depending on the composition of thebioactive agents and the beads. The functionalization of solid supportsurfaces such as certain polymers with chemically reactive groups suchas thiols, amines, carboxyls, etc. is generally known in the art.Accordingly, “blank” microspheres may be used that have surfacechemistries that facilitate the attachment of the desired functionalityby the user. Some examples of these surface chemistries for blankmicrospheres include, but are not limited to, amino groups includingaliphatic and aromatic amines, carboxylic acids, aldehydes, amides,chloromethyl groups, hydrazide, hydroxyl groups, sulfonates andsulfates.

These functional groups can be used to add any number of differentcandidate agents to the beads, generally using known chemistries. Forexample, candidate agents containing carbohydrates may be attached to anamino-functionalized support; the aldehyde of the carbohydrate is madeusing standard techniques, and then the aldehyde is reacted with anamino group on the surface. In an alternative embodiment, a sulfhydryllinker may be used. There are a number of sulfhydryl reactive linkersknown in the art such as SPDP, maleimides, α-haloacetyls, and pyridyldisulfides (see for example the 1994 Pierce Chemical Company catalog,technical section on cross-linkers, pages 155-200, incorporated hereinby reference) which can be used to attach cysteine containingproteinaceous agents to the support. Alternatively, an amino group onthe candidate agent may be used for attachment to an amino group on thesurface. For example, a large number of stable bifunctional groups arewell known in the art, including homobifunctional and heterobifunctionallinkers (see Pierce Catalog and Handbook, pages 155-200). In anadditional embodiment, carboxyl groups (either from the surface or fromthe candidate agent) may be derivatized using well known linkers (seethe Pierce catalog). For example, carbodiimides activate carboxyl groupsfor attack by good nucleophiles such as amines (see Torchilin et al.,Critical Rev. Therapeutic Drug Carrier Systems, 7(4):275-308 (1991),expressly incorporated herein). Proteinaceous candidate agents may alsobe attached using other techniques known in the art, for example for theattachment of antibodies to polymers; see Slinkin et al., Bioconj. Chem.2:342-348 (1991); Torchilin et al., supra; Trubetskoy et al., Bioconj.Chem. 3:323-327 (1992); King et al., Cancer Res. 54:6176-6185 (1994);and Wilbur et al., Bioconjugate Chem. 5:220-235 (1994), all of which arehereby expressly incorporated by reference). It should be understoodthat the candidate agents may be attached in a variety of ways,including those listed above. What is important is that manner ofattachment does not significantly alter the functionality of thecandidate agent; that is, the candidate agent should be attached in sucha flexible manner as to allow its interaction with a target.

Specific techniques for immobilizing enzymes on microspheres are knownin the prior art. In one case, NH₂ surface chemistry microspheres areused. Surface activation is achieved with a 2.5% glutaraldehyde inphosphate buffered saline (10 mM) providing a pH of 6.9. (138 mM NaCl,2.7 mM, KCl). This is stirred on a stir bed for approximately 2 hours atroom temperature. The microspheres are then rinsed with ultrapure waterplus 0.01% tween 20 (surfactant)-0.02%, and rinsed again with a pH 7.7PBS plus 0.01% tween 20. Finally, the enzyme is added to the solution,preferably after being prefiltered using a 0.45 μm amicon micropurefilter.

In some embodiments, the beads may additionally comprise an opticalsignature, that can be used to identify the bioactive agent; see forexample PCT US98/05025, PCT/US99/20914, U.S. Pat. No. 6,023,540 and U.S.Ser. Nos. 09/151,877 and 09/450,829, all of which are expresslyincorporated herein by reference.

In some embodiments, the microspheres may additionally compriseidentifier binding ligands for use in certain decoding systems. By“identifier binding ligands” or “IBLs” herein is meant a compound thatwill specifically bind a corresponding decoder binding ligand (DBL) tofacilitate the elucidation of the identity of the bioactive agentattached to the bead. That is, the IBL and the corresponding DBL form abinding partner pair. By “specifically bind” herein is meant that theIBL binds its DBL with specificity sufficient to differentiate betweenthe corresponding DBL and other DBLs (that is, DBLs for other IBLs), orother components or contaminants of the system. The binding should besufficient to remain bound under the conditions of the decoding step,including wash steps to remove non-specific binding. In someembodiments, for example when the IBLs and corresponding DBLs areproteins or nucleic acids, the dissociation constants of the IBL to itsDBL will be less than about 10⁻⁴-10⁻⁶ M⁻¹, with less than about 10⁻⁵ to10⁻⁹ M⁻¹ being preferred and less than about 10⁻⁷-10⁻⁹ M⁻¹ beingparticularly preferred.

IBL-DBL binding pairs are known or can be readily found using knowntechniques. For example, when the IBL is a protein, the DBLs includeproteins (particularly including antibodies or fragments thereof (FAbs,etc.)) or small molecules, or vice versa (the IBL is an antibody and theDBL is a protein). Metal ion—metal ion ligands or chelators pairs arealso useful. Antigen-antibody pairs, enzymes and substrates orinhibitors, other protein-protein interacting pairs, receptor-ligands,complementary nucleic acids, and carbohydrates and their bindingpartners are also suitable binding pairs. Nucleic acid—nucleic acidbinding proteins pairs are also useful. Similarly, as is generallydescribed in U.S. Pat. Nos. 5,270,163, 5,475,096, 5,567,588, 5,595,877,5,637,459, 5,683,867, 5,705,337, and related patents, herebyincorporated by reference, nucleic acid “aptamers” can be developed forbinding to virtually any target; such an aptamer-target pair can be usedas the IBL-DBL pair. Similarly, there is a wide body of literaturerelating to the development of binding pairs based on combinatorialchemistry methods.

In a preferred embodiment, the IBL is a molecule whose color orluminescence properties change in the presence of a selectively-bindingDBL. For example, the IBL may be a fluorescent pH indicator whoseemission intensity changes with pH. Similarly, the IBL may be afluorescent ion indicator, whose emission properties change with ionconcentration.

Alternatively, the IBL is a molecule whose color or luminescenceproperties change in the presence of various solvents. For example, theIBL may be a fluorescent molecule such as an ethidium salt whosefluorescence intensity increases in hydrophobic environments. Similarly,the IBL may be a derivative of fluorescein whose color changes betweenaqueous and nonpolar solvents.

In one embodiment, the DBL may be attached to a bead, i.e. a “decoderbead”, that may carry a label such as a fluorophore.

In a preferred embodiment, the IBL-DBL pair comprise substantiallycomplementary single-stranded nucleic acids. In this embodiment, thebinding ligands can be referred to as “identifier probes” and “decoderprobes”. Generally, the identifier and decoder probes range from about 4basepairs in length to about 1000, with from about 6 to about 100 beingpreferred, and from about 8 to about 40 being particularly preferred.What is important is that the probes are long enough to be specific,i.e. to distinguish between different IBL-DBL pairs, yet short enough toallow both a) dissociation, if necessary, under suitable experimentalconditions, and b) efficient hybridization.

In a preferred embodiment, as is more fully outlined below, the IBLs donot bind to DBLs. Rather, the IBLs are used as identifier moieties(“IMs”) that are identified directly, for example through the use ofmass spectroscopy.

In a preferred embodiment, the microspheres do not contain an opticalsignature. That is, as outlined in PCT US98/05025, PCT/US99/20914, U.S.Pat. No. 6,023,540 and U.S. Ser. Nos. 09/151,877 and 09/450,829, eachsubpopulation of microspheres may comprise a unique optical signature oroptical tag that is used to identify the unique bioactive agent of thatsubpopulation of microspheres; that is, decoding utilizes opticalproperties of the beads such that a bead comprising the unique opticalsignature may be distinguished from beads at other locations withdifferent optical signatures. This assigns each bioactive agent a uniqueoptical signature such that any microspheres comprising that bioactiveagent are identifiable on the basis of the signature. These opticalsignatures comprised dyes, usually chromophores or fluorophores, thatwere entrapped or attached to the beads themselves. Diversity of opticalsignatures utilized different fluorochromes, different ratios ofmixtures of fluorochromes, and different concentrations (intensities) offluorochromes.

In a preferred embodiment, the arrays do rely solely on the use ofoptical properties to decode the arrays. However, as will be appreciatedby those in the art, it is possible in some embodiments to utilizeoptical signatures as an additional coding method, in conjunction withthe other methods outlined below. Thus, for example, as is more fullyoutlined below, the size of the array may be effectively increased whileusing a single set of decoding moieties in several ways, one of which isthe use of optical signatures one some beads. Thus, for example, usingone “set” of decoding molecules, the use of two populations of beads,one with an optical signature and one without, allows the effectivedoubling of the array size. The use of multiple optical signaturessimilarly increases the possible size of the array.

In a preferred embodiment, each subpopulation of beads comprises aplurality of different IBLs. By using a plurality of different IBLs toencode each bioactive agent, the number of possible unique codes issubstantially increased. That is, by using one unique IBL per bioactiveagent, the size of the array will be the number of unique IBLs (assumingno “reuse” occurs, as outlined below). However, by using a plurality ofdifferent IBLs per bead, n, the size of the array can be increased to2^(n), when the presence or absence of each IBL is used as theindicator. For example, the assignment of 10 IBLs per bead generates a10 bit binary code, where each bit can be designated as “1” (IBL ispresent) or “0” (IBL is absent). A 10 bit binary code has 2¹⁰ possiblevariants. However, as is more fully discussed below, the size of thearray may be further increased if another parameter is included such asconcentration or intensity; thus for example, if two differentconcentrations of the IBL are used, then the array size increases as3^(n). Thus, in this embodiment, each individual bioactive agent in thearray is assigned a combination of IBLs, which can be added to the beadsprior to the addition of the bioactive agent, after, or during thesynthesis of the bioactive agent, i.e. simultaneous addition of IBLs andbioactive agent components.

Alternatively, when the bioactive agent is a polymer of differentresidues, i.e. when the bioactive agent is a protein or nucleic acid,the combination of different IBLs can be used to elucidate the sequenceof the protein or nucleic acid.

Thus, for example, using two different IBLs (IBL 1 and IBL2), the firstposition of a nucleic acid can be elucidated: for example, adenosine canbe represented by the presence of both IBL1 and IBL2; thymidine can berepresented by the presence of IBL1 but not IBL2, cytosine can berepresented by the presence of IBL2 but not IBL1, and guanosine can berepresented by the absence of both. The second position of the nucleicacid can be done in a similar manner using IBL3 and IBL4; thus, thepresence of IBL1, IBL2, IBL3 and IBL4 gives a sequence of AA; IBL1,IBL2, and IBL3 shows the sequence AT; IBL1, IBL3 and IBL4 gives thesequence TA, etc. The third position utilizes IBL5 and IBL6, etc. Inthis way, the use of 20 different identifiers can yield a unique codefor every possible 10-mer.

The system is similar for proteins but requires a larger number ofdifferent IBLs to identify each position, depending on the alloweddiversity at each position. Thus for example, if every amino acid isallowed at every position, five different IBLs are required for eachposition. However, as outlined above, for example when using randompeptides as the bioactive agents, there may be bias built into thesystem; not all amino acids may be present at all positions, and somepositions may be preset; accordingly, it may be possible to utilize fourdifferent IBLs for each amino acid.

In this way, a sort of “bar code” for each sequence can be constructed;the presence or absence of each distinct IBL will allow theidentification of each bioactive agent.

In addition, the use of different concentrations or densities of IBLsallows a “reuse” of sorts. If, for example, the bead comprising a firstagent has a IX concentration of IBL, and a second bead comprising asecond agent has a 10× concentration of IBL, using saturatingconcentrations of the corresponding labelled DBL allows the user todistinguish between the two beads.

Once the microspheres comprising the candidate agents and the uniquetags are generated, they are added to the substrate to form an array. Ingeneral, the methods of making the arrays and of decoding the arrays isdone to maximize the number of different candidate agents that can beuniquely encoded. The compositions of the invention may be made in avariety of ways. In general, the arrays are made by adding a solution orslurry comprising the beads to a surface containing the sites forattachment of the beads. This may be done in a variety of buffers,including aqueous and organic solvents, and mixtures. The solvent canevaporate, and excess beads removed.

In one embodiment the beads or microspheres are contacted with ordistributed on the array through the microfluidic channels. That is, thebeads flow through the channels and are allowed to settle into the wellsof the substrate. Beads can be distributed onto the array either priorto or subsequent to their contacting the sample. A preferred embodimentutilizes contacting the beads and the sample prior to loading the array,coupled with mixing, as this can increase the kinetics of binding. Whenthe beads are contacted with the sample prior to distribution, thesolution may be “emptied” into a microwell array. That is the sampleincluding the beads flows into a channel or detection well thatcomprises microwells. The beads settle into the wells and excess sampleis removed.

In one embodiment the invention provides a method of loading beads intoan array. In a preferred embodiment the invention provides a method forincreasing the filling efficiency of beads on an array. The array maycontain wells as described herein. In one embodiment the inventionincludes the combination of microfluidics and fluid flow, includingelectroosmotic flow, with microsphere arrays. Benefits associated withusing microfluidics to perform bead loading include: 1) overall volumesemployed are drastically reduced which in turn reduces the number ofbeads that need to be synthesized; 2) microchannels can be made toexactly match the size of the array, thus serving to deliver the beadsin a more efficient, focused manner with minimal wastage.

In a first embodiment, an array is incorporated into a bead deliverychannel, i.e. a microchannel, according to one of several methods. Thebead delivery channel may be microfabricated. Most simply, the array,which is preferably a microwell array, is embossed or molded directlyinto the floor at one or more defined locations throughout themicrochannel. In a preferred embodiment the microchannel and array areformed from plastic (which includes a wide scope of polymericmaterials), although the device could also be micromachined into othermaterials such as silicon or glass, for example. The array could also befabricated separately such as in the case of a fiber-optic bundle. Thearray could then be mated to the microfluidics through microcouplers orpress fits through a port in the side wall of a plastic microfluidicpart, for example.

In addition, a particular bead population of interest is introduced intothe microwell array. Although the bead mixture can be prepared ahead oftime manually or with the aid of a robotic dispenser, in someembodiments it is preferable to build the bead mixture on-chip, i.e. onthe array. The use of microfluidics has the advantage of handlingextremely small volumes of solutions with very tight control over thesevolumes. Accordingly, the invention provides improved bead mixing (i.e.more even representation of each bead type in the final mixture) byexploiting these advantages. In one embodiment improved bead mixing isaccomplished by using a microchannel tree structure upstream from thearray (see FIG. 2), containing multiple inlets which gradually convergeinto a single channel. That is, a plurality of channels converge into acommon chamber or module. This chamber or module may be a detectionchamber or module. Alternatively, it may be a chamber for processing, orpreparing the beads.

In some embodiments, reservoirs are present at the mouth of each channelwhere the different bead solutions reside. The bead solutions may beeither manually pipetted or, preferably, robotically dispensed. In apreferred embodiment, the volume in the reservoir is several timeslarger than the amount of solution to be drawn into the channel. Assuch, the uniformity of the final bead pool is not limited by the degreeto which one can uniformly pipette or dispense samples into thechannels. Rather the invention provides the advantage of drawing preciseamounts of suspension, for example, electrokinetically, from eachreservoir. The bead suspensions continue to converge until one mixtureis obtained in the final channel structure.

In additional embodiments, other methods of moving the beads through thechannels could be used, such as capillary action, electrostatic forces,centrifugal force, or simply gravity. In addition, if it was desired tohave beads present in a mixture in specific unequal amounts, the numberof channels devoted to any given bead type could be varied.Alternatively, channels could be made of different lengths orthicknesses in order to effect desired changes in final concentration ofa particular starting bead type. Also, the structure could be designedwith far more (or less) complexity and multiplexing, depending on thepooling needs.

In an additional embodiment, for even more precise control over mixing abead counting device is incorporated into the microchannel mixer. Inthis embodiment each reservoir is triggered successively to flow, i.e.distribute beads, through the network. The beads are counted before thenext bead type is injected. This allows for precise control over exactlyhow many beads of each type are present in the final mixture. In someembodiments, the beads are then distributed into a final “holdingreservoir” until all bead types were present in the mixture. In anadditional embodiment, one could use voltage displacement measurements(Courter principle) or light scattering methods, and build a narrowregion into the device that allows only single beads to pass by thecounting region at a time (see, for example FIG. 3).

In an additional embodiment, the present invention provides analternative approach. Using microchannels and fluid flow, such aselectroosmotic flow, in conjunction with the array, a defined quantityof beads or a bead mixture is delivered to the array region and then thedirection of the bead flow is repeatedly reversed over the array untilevery well or substantially every well is filled. When electroosmoticpumping is used, the method includes alternately changing the potentialsat either end of the microchannel in a continuous fashion. Other methodsof reversing fluid flows can be used depending on the kind of pumpingbeing used. This results in the formation of a type of sifter with nomoving mechanical parts (see, for example, FIG. 4). The repeatedback-and-forth motion of the bead suspension also promotes theimmobilization of only the best-suited (size or otherwise) microspheresto create a highly stable array. In addition the invention includes theformation of charge cross-sections in the channel that promote thetravel of beads along the floor such that their interaction with thewells is enhanced.

In an alternative embodiment, the beads are applied to or distributedonto the array, including fiber optic bundles, prior to combining thearray with the microfluidic chip.

It should be noted that not all sites of an array may comprise a bead;that is, there may be some sites on the substrate surface which areempty. In addition, there may be some sites that contain more than onebead, although this is generally not preferred.

In some embodiments, for example when chemical attachment is done, it ispossible to attach the beads in a non-random or ordered way. Forexample, using photoactivatible attachment linkers or photoactivatibleadhesives or masks, selected sites on the array may be sequentiallyrendered suitable for attachment, such that defined populations of beadsare laid down.

The arrays of the present invention are constructed such thatinformation about the identity of the candidate agent is built into thearray, such that the random deposition of the beads in the fiber wellscan be “decoded” to allow identification of the candidate agent at allpositions. This may be done in a variety of ways, and either before,during or after the use of the array to detect target molecules, as isoutlined in U.S. Ser. Nos. 60/090,473, 09/189,543, 09/344,526 and60/172,106 and PCT/US99/14387, all of which are expressly incorporatedherein by reference.

Thus, after the array is made, it is “decoded” in order to identify thelocation of one or more of the bioactive agents, i.e. each subpopulationof beads, on the substrate surface.

In a preferred embodiment, a selective decoding system is used. In thiscase, only those microspheres exhibiting a change in the optical signalas a result of the binding of a target analyte are decoded. This iscommonly done when the number of “hits”, i.e. the number of sites todecode, is generally low. That is, the array is first scanned underexperimental conditions in the absence of the target analytes. Thesample containing the target analytes is added, and only those locationsexhibiting a change in the optical signal are decoded. For example, thebeads at either the positive or negative signal locations may be eitherselectively tagged or released from the array (for example through theuse of photocleavable linkers), and subsequently sorted or enriched in afluorescence-activated cell sorter (FACS). That is, either all thenegative beads are released, and then the positive beads are eitherreleased or analyzed in situ, or alternatively all the positives arereleased and analyzed. Alternatively, the labels may comprisehalogenated aromatic compounds, and detection of the label is done usingfor example gas chromatography, chemical tags, isotopic tags massspectral tags.

As will be appreciated by those in the art, this may also be done insystems where the array is not decoded; i.e. there need not ever be acorrelation of bead composition with location. In this embodiment, thebeads are loaded on the array, and the assay is run. The “positives”,i.e. those beads displaying a change in the optical signal as is morefully outlined below, are then “marked” to distinguish or separate themfrom the “negative” beads. This can be done in several ways, preferablyusing fiber optic arrays. In a preferred embodiment, each bead containsa fluorescent dye. After the assay and the identification of the“positives” or “active beads”, light is shown down either only thepositive fibers or only the negative fibers, generally in the presenceof a light-activated reagent (typically dissolved oxygen). In the formercase, all the active beads are photobleached. Thus, upon non-selectiverelease of all the beads with subsequent sorting, for example using afluorescence activated cell sorter (FACS) machine, the non-fluorescentactive beads can be sorted from the fluorescent negative beads.Alternatively, when light is shown down the negative fibers, all thenegatives are non-fluorescent and the positives are fluorescent, andsorting can proceed. The characterization of the attached bioactiveagent may be done directly, for example using mass spectroscopy.

Alternatively, the identification may occur through the use ofidentifier moieties (“IMs”), which are similar to IBLs but need notnecessarily bind to DBLs. That is, rather than elucidate the structureof the bioactive agent directly, the composition of the IMs may serve asthe identifier. Thus, for example, a specific combination of IMs canserve to code the bead, and be used to identify the agent on the beadupon release from the bead followed by subsequent analysis, for exampleusing a gas chromatograph or mass spectroscope.

Alternatively, rather than having each bead contain a fluorescent dye,each bead comprises a non-fluorescent precursor to a fluorescent dye.For example, using photocleavable protecting groups, such as certainortho-nitrobenzyl groups, on a fluorescent molecule, photoactivation ofthe fluorochrome can be done. After the assay, light is shown down againeither the “positive” or the “negative” fibers, to distinguish thesepopulations. The illuminated precursors are then chemically converted toa fluorescent dye. All the beads are then released from the array, withsorting, to form populations of fluorescent and non-fluorescent beads(either the positives and the negatives or vice versa).

In an alternate preferred embodiment, the sites of attachment of thebeads (for example the wells) include a photopolymerizable reagent, orthe photopolymerizable agent is added to the assembled array. After thetest assay is run, light is shown down again either the “positive” orthe “negative” fibers, to distinguish these populations. As a result ofthe irradiation, either all the positives or all the negatives arepolymerized and trapped or bound to the sites, while the otherpopulation of beads can be released from the array.

In a preferred embodiment, the location of every bioactive agent isdetermined using decoder binding ligands (DBLs). As outlined above, DBLsare binding ligands that will either bind to identifier binding ligands,if present, or to the bioactive agents themselves, preferably when thebioactive agent is a nucleic acid or protein.

In a preferred embodiment, as outlined above, the DBL binds to the IBL.

In a preferred embodiment, the bioactive agents are single-strandednucleic acids and the DBL is a substantially complementarysingle-stranded nucleic acid that binds (hybridizes) to the bioactiveagent, termed a decoder probe herein. A decoder probe that issubstantially complementary to each candidate probe is made and used todecode the array. In this embodiment, the candidate probes and thedecoder probes should be of sufficient length (and the decoding step rununder suitable conditions) to allow specificity; i.e. each candidateprobe binds to its corresponding decoder probe with sufficientspecificity to allow the distinction of each candidate probe.

In a preferred embodiment, the DBLs are either directly or indirectlylabeled. By “labeled” herein is meant that a compound has at least oneelement, isotope or chemical compound attached to enable the detectionof the compound. In general, labels fall into three classes: a) isotopiclabels, which may be radioactive or heavy isotopes; b) magnetic,electrical, thermal; and c) colored or luminescent dyes; although labelsinclude enzymes and particles such as magnetic particles as well.Preferred labels include luminescent labels, including fluorochromes. Ina preferred embodiment, the DBL is directly labeled, that is, the DBLcomprises a label. In an alternate embodiment, the DBL is indirectlylabeled; that is, a labeling binding ligand (LBL) that will bind to theDBL is used. In this embodiment, the labeling binding ligand-DBL paircan be as described above for IBL-DBL pairs.

Accordingly, the identification of the location of the individual beads(or subpopulations of beads) is done using one or more decoding stepscomprising a binding between the labeled DBL and either the IBL or thebioactive agent (i.e. a hybridization between the candidate probe andthe decoder probe when the bioactive agent is a nucleic acid). Afterdecoding, the DBLs can be removed and the array can be used; however, insome circumstances, for example when the DBL binds to an IBL and not tothe bioactive agent, the removal of the DBL is not required (although itmay be desirable in some circumstances). In addition, as outlinedherein, decoding may be done either before the array is used to in anassay, during the assay, or after the assay.

In one embodiment, a single decoding step is done. In this embodiment,each DBL is labeled with a unique label, such that the number of uniquetags is equal to or greater than the number of bioactive agents(although in some cases, “reuse” of the unique labels can be done, asdescribed herein; similarly, minor variants of candidate probes canshare the same decoder, if the variants are encoded in anotherdimension, i.e. in the bead size or label). For each bioactive agent orIBL, a DBL is made that will specifically bind to it and contains aunique tag, for example one or more fluorochromes. Thus, the identity ofeach DBL, both its composition (i.e. its sequence when it is a nucleicacid) and its label, is known. Then, by adding the DBLs to the arraycontaining the bioactive agents under conditions which allow theformation of complexes (termed hybridization complexes when thecomponents are nucleic acids) between the DBLs and either the bioactiveagents or the IBLs, the location of each DBL can be elucidated. Thisallows the identification of the location of each bioactive agent; therandom array has been decoded. The DBLs can then be removed, ifnecessary, and the target sample applied.

In a preferred embodiment, the number of unique labels is less than thenumber of unique bioactive agents, and thus a sequential series ofdecoding steps are used. To facilitate the discussion, this embodimentis explained for nucleic acids, although other types of bioactive agentsand DBLs are useful as well. In this embodiment, decoder probes aredivided into n sets for decoding. The number of sets corresponds to thenumber of unique tags. Each decoder probe is labeled in n separatereactions with n distinct tags. All the decoder probes share the same ntags. The decoder probes are pooled so that each pool contains only oneof the n tag versions of each decoder, and no two decoder probes havethe same sequence of tags across all the pools. The number of poolsrequired for this to be true is determined by the number of decoderprobes and the n. Hybridization of each pool to the array generates asignal at every address. The sequential hybridization of each pool inturn will generate a unique, sequence-specific code for each candidateprobe. This identifies the candidate probe at each address in the array.For example, if four tags are used, then 4×n sequential hybridizationscan ideally distinguish 4^(n) sequences, although in some cases moresteps may be required. After the hybridization of each pool, the hybridsare denatured and the decoder probes removed, so that the probes arerendered single-stranded for the next hybridization (although it is alsopossible to hybridize limiting amounts of target so that the availableprobe is not saturated. Sequential hybridizations can be carried out andanalyzed by subtracting pre-existing signal from the previoushybridization).

An example is illustrative. Assuming an array of 16 probe nucleic acids(numbers 1-16), and four unique tags (four different fluors, forexample; labels A-D). Decoder probes 1-16 are made that correspond tothe probes on the beads. The first step is to label decoder probes 1-4with tag A, decoder probes 5-8 with tag B, decoder probes 9-12 with tagC, and decoder probes 13-16 with tag D. The probes are mixed and thepool is contacted with the array containing the beads with the attachedcandidate probes. The location of each tag (and thus each decoder andcandidate probe pair) is then determined. The first set of decoderprobes are then removed. A second set is added, but this time, decoderprobes 1, 5, 9 and 13 are labeled with tag A, decoder probes 2, 6, 10and 14 are labeled with tag B, decoder probes 3, 7, 11 and 15 arelabeled with tag C, and decoder probes 4, 8, 12 and 16 are labeled withtag D. Thus, those beads that contained tag A in both decoding stepscontain candidate probe 1; tag A in the first decoding step and tag B inthe second decoding step contain candidate probe 2; tag A in the firstdecoding step and tag C in the second step contain candidate probe 3;etc.

In one embodiment, the decoder probes are labeled in situ; that is, theyneed not be labeled prior to the decoding reaction. In this embodiment,the incoming decoder probe is shorter than the candidate probe, creatinga 5′ “overhang” on the decoding probe. The addition of labeled ddNTPs(each labeled with a unique tag) and a polymerase will allow theaddition of the tags in a sequence specific manner, thus creating asequence-specific pattern of signals. Similarly, other modifications canbe done, including ligation, etc.

In addition, since the size of the array will be set by the number ofunique decoding binding ligands, it is possible to “reuse” a set ofunique DBLs to allow for a greater number of test sites. This may bedone in several ways; for example, by using some subpopulations thatcomprise optical signatures. Similarly, the use of a positional codingscheme within an array; different sub-bundles may reuse the set of DBLs.Similarly, one embodiment utilizes bead size as a coding modality, thusallowing the reuse of the set of unique DBLs for each bead size.Alternatively, sequential partial loading of arrays with beads can alsoallow the reuse of DBLs. Furthermore, “code sharing” can occur as well.

In a preferred embodiment, the DBLs may be reused by having somesubpopulations of beads comprise optical signatures. In a preferredembodiment, the optical signature is generally a mixture of reporterdyes, preferably fluorescent. By varying both the composition of themixture (i.e. the ratio of one dye to another) and the concentration ofthe dye (leading to differences in signal intensity), matrices of uniqueoptical signatures may be generated. This may be done by covalentlyattaching the dyes to the surface of the beads, or alternatively, byentrapping the dye within the bead. The dyes may be chromophores orphosphors but are preferably fluorescent dyes, which due to their strongsignals provide a good signal-to-noise ratio for decoding. Suitable dyesfor use in the invention include, but are not limited to, fluorescentlanthanide complexes, including those of Europium and Terbium,fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin,coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, LuciferYellow, Cascade Blue™, Texas Red, and others described in the 6thEdition of the Molecular Probes Handbook by Richard P. Haugland, herebyexpressly incorporated by reference.

In a preferred embodiment, the encoding can be accomplished in a ratioof at least two dyes, although more encoding dimensions may be added inthe size of the beads, for example. In addition, the labels aredistinguishable from one another; thus two different labels may comprisedifferent molecules (i.e. two different fluors) or, alternatively, onelabel at two different concentrations or intensity.

In a preferred embodiment, the dyes are covalently attached to thesurface of the beads. This may be done as is generally outlined for theattachment of the bioactive agents, using functional groups on thesurface of the beads. As will be appreciated by those in the art, theseattachments are done to minimize the effect on the dye.

In a preferred embodiment, the dyes are non-covalently associated withthe beads, generally by entrapping the dyes in the pores of the beads.

Additionally, encoding in the ratios of the two or more dyes, ratherthan single dye concentrations, is preferred since it providesinsensitivity to the intensity of light used to interrogate the reporterdye's signature and detector sensitivity.

In a preferred embodiment, a spatial or positional coding system isdone. In this embodiment, there are sub-bundles or subarrays (i.e.portions of the total array) that are utilized. By analogy with thetelephone system, each subarray is an “area code”, that can have thesame tags (i.e. telephone numbers) of other subarrays, that areseparated by virtue of the location of the subarray. Thus, for example,the same unique tags can be reused from bundle to bundle. Thus, the useof 50 unique tags in combination with 100 different subarrays can forman array of 5000 different bioactive agents. In this embodiment, itbecomes important to be able to identify one bundle from another; ingeneral, this is done either manually or through the use of markerbeads, i.e. beads containing unique tags for each subarray.

In alternative embodiments, additional encoding parameters can be added,such as microsphere size. For example, the use of different size beadsmay also allow the reuse of sets of DBLs; that is, it is possible to usemicrospheres of different sizes to expand the encoding dimensions of themicrospheres. Optical fiber arrays can be fabricated containing pixelswith different fiber diameters or cross-sections; alternatively, two ormore fiber optic bundles, each with different cross-sections of theindividual fibers, can be added together to form a larger bundle; or,fiber optic bundles with fiber of the same size cross-sections can beused, but just with different sized beads. With different diameters, thelargest wells can be filled with the largest microspheres and thenmoving onto progressively smaller microspheres in the smaller wellsuntil all size wells are then filled. In this manner, the same dye ratiocould be used to encode microspheres of different sizes therebyexpanding the number of different oligonucleotide sequences or chemicalfunctionalities present in the array. Although outlined for fiber opticsubstrates, this as well as the other methods outlined herein can beused with other substrates and with other attachment modalities as well.

In a preferred embodiment, the coding and decoding is accomplished bysequential loading of the microspheres into the array. As outlined abovefor spatial coding, in this embodiment, the optical signatures can be“reused”. In this embodiment, the library of microspheres eachcomprising a different bioactive agent (or the subpopulations eachcomprise a different bioactive agent), is divided into a plurality ofsublibraries; for example, depending on the size of the desired arrayand the number of unique tags, 10 sublibraries each comprising roughly10% of the total library may be made, with each sublibrary comprisingroughly the same unique tags. Then, the first sublibrary is added to thefiber optic bundle comprising the wells, and the location of eachbioactive agent is determined, generally through the use of DBLs. Thesecond sublibrary is then added, and the location of each bioactiveagent is again determined. The signal in this case will comprise thesignal from the “first” DBL and the “second” DBL; by comparing the twomatrices the location of each bead in each sublibrary can be determined.Similarly, adding the third, fourth, etc. sublibraries sequentially willallow the array to be filled.

In a preferred embodiment, codes can be “shared” in several ways. In afirst embodiment, a single code (i.e. IBL/DBL pair) can be assigned totwo or more agents if the target analytes different sufficiently intheir binding strengths. For example, two nucleic acid probes used in anmRNA quantitation assay can share the same code if the ranges of theirhybridization signal intensities do not overlap. This can occur, forexample, when one of the target sequences is always present at a muchhigher concentration than the other. Alternatively, the two targetsequences might always be present at a similar concentration, but differin hybridization efficiency.

Alternatively, a single code can be assigned to multiple agents if theagents are functionally equivalent. For example, if a set ofoligonucleotide probes are designed with the common purpose of detectingthe presence of a particular gene, then the probes are functionallyequivalent, even though they may differ in sequence. Similarly, ifclasses of analytes are desired, all probes for different members of aclass such as kinases or G-protein coupled receptors could share a code.Similarly, an array of this type could be used to detect homologs ofknown genes. In this embodiment, each gene is represented by aheterologous set of probes, hybridizing to different regions of the gene(and therefore differing in sequence). The set of probes share a commoncode. If a homolog is present, it might hybridize to some but not all ofthe probes. The level of homology might be indicated by the fraction ofprobes hybridizing, as well as the average hybridization intensity.Similarly, multiple antibodies to the same protein could all share thesame code.

As will be appreciated by those in the art, the decoding may be doneprior to the placement of the detection module in the microfluidicdevice, or afterwards.

Once made, the compositions of the invention find use in a number ofapplications. In a preferred embodiment, the compositions are used toprobe a sample solution for the presence or absence of a target analyte,including the quantification of the amount of target analyte present, asdefined above.

In a preferred embodiment, the target analyte is a nucleic acid. Theseassays find use in a wide variety of applications.

In a preferred embodiment, the probes are used in genetic diagnosis. Forexample, probes can be made using the techniques disclosed herein todetect target sequences such as the gene for nonpolyposis colon cancer,the BRCA1 breast cancer gene, P53, which is a gene associated with avariety of cancers, the Apo E4 gene that indicates a greater risk ofAlzheimer's disease, allowing for easy presymptomatic screening ofpatients, mutations in the cystic fibrosis gene, cytochrome p450s or anyof the others well known in the art.

In an additional embodiment, viral and bacterial detection is done usingthe complexes of the invention. In this embodiment, probes are designedto detect target sequences from a variety of bacteria and viruses. Forexample, current blood-screening techniques rely on the detection ofanti-HIV antibodies. The methods disclosed herein allow for directscreening of clinical samples to detect HIV nucleic acid sequences,particularly highly conserved HIV sequences. In addition, this allowsdirect monitoring of circulating virus within a patient as an improvedmethod of assessing the efficacy of anti-viral therapies. Similarly,viruses associated with leukemia, HTLV-I and HTLV-II, may be detected inthis way. Bacterial infections such as tuberculosis, chlamydia and othersexually transmitted diseases, may also be detected.

In a preferred embodiment, the nucleic acids of the invention find useas probes for toxic bacteria in the screening of water and food samples.For example, samples may be treated to lyse the bacteria to release itsnucleic acid, and then probes designed to recognize bacterial strains,including, but not limited to, such pathogenic strains as, Salmonella,Campylobacter, Vibrio cholerae, Leishmania, enterotoxic strains of E.coli, and Legionnaire's disease bacteria. Similarly, bioremediationstrategies may be evaluated using the compositions of the invention.

In a further embodiment, the probes are used for forensic “DNAfingerprinting” to match crime-scene DNA against samples taken fromvictims and suspects.

In an additional embodiment, the probes in an array are used forsequencing by hybridization.

The present invention also finds use as a methodology for the detectionof mutations or mismatches in target nucleic acid sequences. Forexample, recent focus has been on the analysis of the relationshipbetween genetic variation and phenotype by making use of polymorphic DNAmarkers. Previous work utilized short tandem repeats (STRs) aspolymorphic positional markers; however, recent focus is on the use ofsingle nucleotide polymorphisms (SNPs), which occur at an averagefrequency of more than 1 per kilobase in human genomic DNA. Some SNPs,particularly those in and around coding sequences, are likely to be thedirect cause of therapeutically relevant phenotypic variants. There area number of well known polymorphisms that cause clinically importantphenotypes; for example, the apoE2/3/4 variants are associated withdifferent relative risk of Alzheimer's and other diseases (see Cordor etal., Science 261(1993). Multiplex PCR amplification of SNP loci withsubsequent hybridization to oligonucleotide arrays has been shown to bean accurate and reliable method of simultaneously genotyping at leasthundreds of SNPs; see Wang et al., Science, 280:1077 (1998); see alsoSchafer et al., Nature Biotechnology 16:33-39 (1998). The compositionsof the present invention may easily be substituted for the arrays of theprior art.

In a preferred embodiment, the compositions of the invention are used toscreen bioactive agents to find an agent that will bind, and preferablymodify the function of, a target molecule. As above, a wide variety ofdifferent assay formats may be run, as will be appreciated by those inthe art. Generally, the target analyte for which a binding partner isdesired is labeled; binding of the target analyte by the bioactive agentresults in the recruitment of the label to the bead, with subsequentdetection.

Generally, a sample containing a target analyte (whether for detectionof the target analyte or screening for binding partners of the targetanalyte) is added to the array, under conditions suitable for binding ofthe target analyte to at least one of the bioactive agents, i.e.generally physiological conditions. The presence or absence of thetarget analyte is then detected. As will be appreciated by those in theart, this may be done in a variety of ways, generally through the use ofa change in an optical signal. This change can occur via many differentmechanisms. A few examples include the binding of a dye-tagged analyteto the bead, the production of a dye species on or near the beads, thedestruction of an existing dye species, a change in the opticalsignature upon analyte interaction with dye on bead, or any otheroptical interrogatable event.

In a preferred embodiment, the change in optical signal occurs as aresult of the binding of a target analyte that is labeled, eitherdirectly or indirectly, with a detectable label, preferably an opticallabel such as a fluorochrome. Thus, for example, when a proteinaceoustarget analyte is used, it may be either directly labeled with a fluor,or indirectly, for example through the use of a labeled antibody.Similarly, nucleic acids are easily labeled with fluorochromes, forexample during PCR amplification as is known in the art. Alternatively,upon binding of the target sequences, a hybridization indicator may beused as the label. Hybridization indicators preferentially associatewith double stranded nucleic acid, usually reversibly. Hybridizationindicators include intercalators and minor and/or major groove bindingmoieties. In a preferred embodiment, intercalators may be used; sinceintercalation generally only occurs in the presence of double strandednucleic acid, only in the presence of target hybridization will thelabel light up. Thus, upon binding of the target analyte to a bioactiveagent, there is a new optical signal generated at that site, which thenmay be detected.

Alternatively, in some cases, as discussed above, the target analytesuch as an enzyme generates a species that is either directly orindirectly optical detectable.

Furthermore, in some embodiments, a change in the optical signature maybe the basis of the optical signal. For example, the interaction of somechemical target analytes with some fluorescent dyes on the beads mayalter the optical signature, thus generating a different optical signal.

As will be appreciated by those in the art, in some embodiments, thepresence or absence of the target analyte may be done using changes inother optical or non-optical signals, including, but not limited to,surface enhanced Raman spectroscopy, surface plasmon resonance,radioactivity, etc.

The assays may be run under a variety of experimental conditions, aswill be appreciated by those in the art. A variety of other reagents maybe included in the screening assays. These include reagents like salts,neutral proteins, e.g. albumin, detergents, etc which may be used tofacilitate optimal protein-protein binding and/or reduce non-specific orbackground interactions. Also reagents that otherwise improve theefficiency of the assay, such as protease inhibitors, nucleaseinhibitors, anti-microbial agents, etc., may be used. The mixture ofcomponents may be added in any order that provides for the requisitebinding. Various blocking and washing steps may be utilized as is knownin the art.

In a preferred embodiment, two-color competitive hybridization assaysare run. These assays can be based on traditional sandwich assays. Thebeads contain a capture sequence located on one side (upstream ordownstream) of the SNP, to capture the target sequence. Two SNPallele-specific probes, each labeled with a different fluorophor, arehybridized to the target sequence. The genotype can be obtained from aratio of the two signals, with the correct sequence generally exhibitingbetter binding. This has an advantage in that the target sequence itselfneed not be labeled. In addition, since the probes are competing, thismeans that the conditions for binding need not be optimized. Underconditions where a mismatched probe would be stably bound, a matchedprobe can still displace it. Therefore the competitive assay can providebetter discrimination under those conditions. Because many assays arecarried out in parallel, conditions cannot be optimized for every probesimultaneously. Therefore, a competitive assay system can be used tohelp compensate for non-optimal conditions for mismatch discrimination.

In a preferred embodiment, dideoxynucleotide chain-terminationsequencing is done using the compositions of the invention. In thisembodiment, a DNA polymerase is used to extend a primer usingfluorescently labeled ddNTPs. The 3′ end of the primer is locatedadjacent to the SNP site. In this way, the single base extension iscomplementary to the sequence at the SNP site. By using four differentfluorophores, one for each base, the sequence of the SNP can be deducedby comparing the four base-specific signals. This may be done in severalways. In a first embodiment, the capture probe can be extended; in thisapproach, the probe must either be synthesized 5′-3′ on the bead, orattached at the 5′ end, to provide a free 3′ end for polymeraseextension. Alternatively, a sandwich type assay can be used; in thisembodiment, the target is captured on the bead by a probe, then a primeris annealed and extended. Again, in the latter case, the target sequenceneed not be labeled. In addition, since sandwich assays require twospecific interactions, this provides increased stringency which isparticularly helpful for the analysis of complex samples.

SNP analysis may also be done using pyrosequencing and other methods, asis generally described in U.S. Ser. Nos. 60/130,089, 60/160,027,09/513,362, 60/135,051, 60/161,148, 09/517,945, 60/135,053, 09/425,633,09/535,854, 09/553,993 and 09/556,463, and PCT application entitled“Detection of Nucleic Acid Reactions on Bead Arrays” filed Apr. 20, 2000(no serial number received) expressly incorporated herein by reference.

In some embodiments, the use of adapters as described in U.S. Ser. Nos.60/135,123 and 60/160,917, both of which are expressly incorporatedherein by reference, finds use in the invention.

In addition, when the target analyte and the DBL both bind to the agent,it is also possible to do detection of non-labeled target analytes viacompetition of decoding.

In a preferred embodiment, the methods of the invention are useful inarray quality control. Prior to this invention, no methods have beendescribed that provide a positive test of the performance of every probeon every array. Decoding of the array not only provides this test, italso does so by making use of the data generated during the decodingprocess itself. Therefore, no additional experimental work is required.The invention requires only a set of data analysis algorithms that canbe encoded in software.

The quality control procedure can identify a wide variety of systematicand random problems in an array. For example, random specks of dust orother contaminants might cause some sensors to give an incorrectsignal—this can be detected during decoding. The omission of one or moreagents from multiple arrays can also be detected. An advantage of thisquality control procedure is that it can be implemented immediated priorto the assay itself, and is a true functional test of each individualsensor. Therefore any problems that might occur between array assemblyand actual use can be detected. In applications where a very high levelof confidence is required, and/or there is a significant chance ofsensor failure during the experimental procedure, decoding and qualitycontrol can be conducted both before and after the actual sampleanalysis.

In a preferred embodiment, the arrays can be used to do reagent qualitycontrol. In many instances, biological macromolecules are used asreagents and must be quality controlled. For example, large sets ofoligonucleotide probes may be provided as reagents. It is typicallydifficult to perform quality control on large numbers of differentbiological macromolecules. The approach described here can be used to dothis by treating the reagents (formulated as the DBLs) as variableinstead of the arrays.

In a preferred embodiment, the methods outlined herein are used in arraycalibration. For many applications, such as mRNA quantitation, it isdesirable to have a signal that is a linear response to theconcentration of the target analyte, or, alternatively, if non-linear,to determine a relationship between concentration and signal, so thatthe concentration of the target analyte can be estimated. Accordingly,the present invention provides methods of creating calibration curves inparallel for multiple beads in an array. The calibration curves can becreated under conditions that simulate the complexity of the sample tobe analyzed. Each curve can be constructed independently of the others(e.g. for a different range of concentrations), but at the same time asall the other curves for the array. Thus, in this embodiment, thesequential decoding scheme is implemented with different concentrationsbeing used as the code “labels”, rather than different fluorophores. Inthis way, signal as a response to concentration can be measured for eachbead. This calibration can be carried out just prior to array use, sothat every probe on every array is individually calibrated as needed.

In a preferred embodiment, the methods of the invention can be used inassay development as well. Thus, for example, the methods allow theidentification of good and bad probes; as is understood by those in theart, some probes do not function well because they do not hybridizewell, or because they cross-hybridize with more than one sequence. Theseproblems are easily detected during decoding. The ability to rapidlyassess probe performance has the potential to greatly reduce the timeand expense of assay development.

Similarly, in a preferred embodiment, the methods of the invention areuseful in quantitation in assay development. Major challenges of manyassays is the ability to detect differences in analyte concentrationsbetween samples, the ability to quantitate these differences, and tomeasure absolute concentrations of analytes, all in the presence of acomplex mixture of related analytes. An example of this problem is thequantitation of a specific mRNA in the presence of total cellular mRNA.One approach that has been developed as a basis of mRNA quantitationmakes use of a multiple match and mismatch probe pairs (Lockhart et al.,1996), hereby incorporated by reference in its entirety. While thisapproach is simple, it requires relatively large numbers of probes. Inthis approach, a quantitative response to concentration is obtained byaveraging the signals from a set of different probes to the gene orsequence of interest. This is necessary because only some probes respondquantitatively, and it is not possible to predict these probes withcertainty. In the absence of prior knowledge, only the average responseof an appropriately chosen collection of probes is quantitative.However, in the present invention, this can be applied generally tonucleic acid based assays as well as other assays. In essence, theapproach is to identify the probes that respond quantitatively in aparticular assay, rather than average them with other probes. This isdone using the array calibration scheme outlined above, in whichconcentration-based codes are used. Advantages of this approach include:fewer probes are needed; the accuracy of the measurement is lessdependent on the number of probes used; and that the response of thesensors is known with a high level of certainty, since each and everysequence can be tested in an efficient manner. It is important to notethat probes that perform well are chosen empirically, which avoids thedifficulties and uncertainties of predicting probe performance,particularly in complex sequence mixtures. In contrast, in experimentsdescribed to date with ordered arrays, relatively small numbers ofsequences are checked by performing quantitative spiking experiments, inwhich a known mRNA is added to a mixture.

Generally, the methods are as follows. In a preferred embodiment, thetarget is moved into the detection module. In general, two methods maybe employed; the assay complexes as described below are formed first(i.e. all the soluble components are added together, eithersimultaneously or sequentially), “upstream” of the detection module, andthen the complex is added to the surface for subsequent binding to adetection array. Alternatively, the target may be added where it bindsthe capture binding ligand and then additional components are added. Thelatter is described in detail below, but either procedure may befollowed. Similarly, some components may be added, electrophoresed, andother components added; for example, the target analyte may be combinedwith any capture extender probes and then transported, etc. In addition,as outlined herein, “washing” steps may be done using the introductionof buffer into the detection module, wherein excess reagents (non-boundanalytes, excess probes, etc.) can be driven from the surface.

In a preferred embodiment, the methods include processing the sampleupstream of the detection chamber. That is, the sample processing occursin one or more channels or chambers of the chip. In one embodiment,sample preparation occurs in more than one channel; however, sampleprocessing occurs in parallel. The prepared sample is then recombinedinto a single channel that flows to the detection module. Thus, forexample, a variety of different PCR reactions may be done in a pluralityof chambers, with all the reaction products being added to a singlearray. Alternatively, parallel reactions can be added to differentarrays. In a preferred embodiment, the reactions happen sequentially;for example, a first PCR reaction can be performed in a first chamberand a “nested” PCR reaction performed in a subsequent chamber.

Sample movement within the channels can occur through conventionalmethods including electro-osmotic flow, capillary action or pressure, asoutlined herein, and includes the use of “on chip” and “off chip” pumps.In one embodiment, movement of the sample stops once the sample contactsthe detection module. This allows time for any of the above-describedassays to occur. Alternatively, movement is not necessarily stopped, butrather slows down as the sample crosses the array. Alternatively, forfast reactions or when recirculation is used, the flow is unchanged.

Regulating sample flow is accomplished by reducing the driving forcethat is applied to the sample. Alternatively, physical aspects of thedetection module can be altered to affect sample flow. In one embodimentthe diameter of the detection module is increased relative to otherchannels. This results in slowing the sample flow.

In an alternative embodiment, sample flow can be re-circulated acrossthe detection module. In this embodiment, a closed looped channel isused to re-circulate the sample. Recirculation also may improve theassay and/or signal detection by facilitating mixing across the array.

The sample is introduced to the array in the detection module, and thenimmobilized or attached to the beads. In one embodiment, this is done byforming an attachment complex (frequently referred to herein as ahybridization complex when nucleic acid components are used) between acapture probe and a portion of the target analyte. Alternatively, theattachment of the target sequence to the beads is done simultaneouslywith the other reactions.

The method proceeds with the introduction of amplifier probes, ifutilized. In a preferred embodiment, the amplifier probe comprises afirst probe sequence that is substantially complementary to a portion ofthe target sequence, and at least one amplification sequence.

In one embodiment, the first probe sequence of the amplifier probe ishybridized to the target sequence, and any unhybridized amplifier probeis removed. This will generally be done as is known in the art, anddepends on the type of assay. When the target sequence is immobilized onthe array surface, the removal of excess reagents generally is done viaone or more washing steps, as will be appreciated by those in the art.

The invention thus provides assay complexes that minimally comprise atarget sequence and a label probe. “Assay complex” herein is meant thecollection of attachment or hybridization complexes comprising analytes,including binding ligands and targets, that allows detection. Thecomposition of the assay complex depends on the use of the differentprobe component outlined herein. The assay complexes may include thetarget sequence, label probes, capture extender probes, label extenderprobes, and amplifier probes, as outlined herein, depending on theconfiguration used.

The assays are generally run under stringency conditions which allowsformation of the label probe attachment complex only in the presence oftarget. Stringency can be controlled by altering a step parameter thatis a thermodynamic variable, including, but not limited to, temperature,formamide concentration, salt concentration, chaotropic saltconcentration pH, organic solvent concentration, etc. Stringency mayalso include the use of an electrophoretic step to drive non-specific(i.e. low stringency) materials away from the detection array.

These parameters may also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus it may be desirableto perform certain steps at higher stringency conditions; for example,when an initial hybridization step is done between the target sequenceand the label extender and capture extender probes. Running this step atconditions which favor specific binding can allow the reduction ofnon-specific binding.

Once the assay complexes are formed on the detection array, detectionproceeds, generally through optical detection of fluorescence. Thus,preferred embodiments utilize detection modules that comprise opticalwindows to allow detection of target analytes.

In a preferred embodiment, mixing of the sample is performed tofacilitate signal detection. That is, as demonstrated in FIG. 1,substantial improvement in signals is observed when sample vibration isimplemented during an experiment. In one embodiment, this vibration ormixing is caused by vibration of the chip itself. Alternatively, themixing is caused by continuous sample flow over the array surface. Inthis embodiment, the flow of the sample over the surface comprisingmicrospheres provides sufficient high aspect ratio features to induce alevel of turbulent flow that enhances the interaction of the sample withthe beads. In an alternative embodiment, the vertical microstructures orposts as described above serve to disrupt the laminar flow over thebeaded surface.

Accordingly, the present invention further provides devices or apparatusfor the detection of analytes using the compositions of the invention.As will be appreciated by those in the art, the modules of the inventioncan be configured in a variety of ways, depending on the number and sizeof samples, and the number and type of desired manipulations.

In a preferred embodiment, when a fiber optic bundle is used in thedetection module, the results from the experiment are read from the endof the bundle not attached to the chip. In this embodiment, this end ofthe bundle is connected a CCD camera or other scanning instrument as isknown in the art. In addition, the results are examined by focusing aconfocal scanning instrument onto the end of the fiber bundle that iswithin the chip.

As outlined herein, the devices of the invention can be used incombination with apparatus for delivering and receiving fluids to andfrom the devices. The apparatus can include a “nesting site” forplacement of the device(s) to hold them in place and for registeringinlet and outlet ports, if present. The apparatus may also include pumps(“off chip pumps”), and means for viewing the contents of the devices,including microscopes, cameras (including CCD cameras and scanners),etc. The apparatus may include electrical contacts in the nesting regionwhich mate with contacts integrated into the structure of the chip, topower heating or electrophoresis, for example. The apparatus may beprovided with conventional circuitry sensors in communication withsensors in the device for thermal regulation, for example for PCRthermal regulation. The apparatus may also include a computer systemcomprising a microprocessor for control of the various modules of thesystem as well as for data analysis.

All references cited herein are incorporated by reference in theirentirety.

1. A microfluidic device for the detection of target analytescomprising: a) a sample inlet port; b) a detection inlet port; c) asubstrate comprising: i) at least one sample handling well; ii) a firstmicrochannel to allow fluid contact between said sample inlet port andsaid sample handling well; iii) a detection module comprising: (a) aplurality of discrete sites in said substrate; and (b) a population ofmicrospheres comprising at least a first and a second subpopulation,wherein each subpopulation comprises a bioactive agent;  wherein saidmicrospheres are distributed on said discrete sites, wherein eachdiscrete site contains not more than one microsphere; and iv) a secondmicrochannel to allow fluid contact between said sample handling welland said detection inlet port.
 2. A microfluidic device comprising asubstrate comprising: a) a plurality of sample handling wells; b) adetection channel comprising: i) a plurality of discrete sites; and ii)a population of microspheres comprising at least a first and a secondsubpopulation, wherein each subpopulation comprises a bioactive agent,wherein said microspheres are distributed on said discrete sites; and c)at least one microchannel to allow fluid contact between each of saidsample handling wells and said detection channel.
 3. The deviceaccording to claim 1 or claim 2, wherein said substrate comprises afiber optic bundle.
 4. The device according to claim 1 or claim 2,wherein each subpopulation further comprises an optical signature. 5.The device according to claim 1 or claim 2, wherein each subpopulationfurther comprises an identifier binding ligand that will bind a decoderbinding ligand such that the identification of the bioactive agent canbe elucidated.
 6. The device according to claim 1 or claim 2, whereinsaid bioactive agent is a nucleic acid.
 7. The device according to claim1 or claim 2, wherein said substrate comprises vertical microstructuresbetween said discrete sites.
 8. The device according to claim 1 or claim2, further comprising a device for regulating sample flow.
 9. The deviceaccording to claim 8, wherein said device comprises a closed loopedchannel.
 10. The device according to claim 1 or claim 2, wherein eachsubpopulation further comprises a decoder binding ligand bound to saidbioactive agent.
 11. A method of assembling a detector in a microfluidicdevice comprising: a) providing a microfluidic device comprising: i) afirst microchannel to allow fluid contact between a sample inlet portand a sample handling well; ii) a second microchannel to allow fluidcontact between said sample handling well and a detection inlet port;and ii) a detection module comprising a substrate with a surfacecomprising discrete sites; b) flowing a fluid across said substrate,said fluid comprising a population of microspheres comprising at least afirst and a second subpopulation, wherein each subpopulation comprises abioactive agent, whereby said beads flow across said discrete sites, andare deposited randomly in said discrete sites; and c) reversing the flowof said fluid.
 12. The method according to claim 11, wherein saidmicrofluidic device further comprises a pump to pump said fluid.
 13. Themethod according to claim 12, wherein said pump is selected from thegroup consisting of an electrohydrodynamic pump, electrokinetic pump andelectroosmotic pump.
 14. A method of assembling a detector in amicrofluidic device comprising: a) providing a microfluidic devicecomprising: i) a plurality of first micro channels; ii) a population ofmicrospheres in each of said microchannels; and iii) at least onereceiving chamber connected to said microchannels. b) flowing saidmicrospheres through said microchannels into said receiving chamber. 15.The method according to claim 14, wherein said receiving chamber is adetection module.
 16. The method according to claim 15, wherein saiddetection module comprises a substrate with a surface comprisingdiscrete sites.
 17. The method according to claim 16, wherein saidpopulation of microspheres is randomly distributed on said substrate.18. The method according to claim 14, wherein said population comprisesa first and second subpopulation whereby said first and secondsubpopulations of microspheres are in said first and secondmicrochannels.